Molecular And Cellular Biology

Question 1

What does cell theory state?

Answer 1

All living organisms are made up of cells (basic structural subunit)
All cells derived from pre existing cells

Question 2

What is the role of the plasma membrane of a cell?

Answer 2

Provides a physical barrier between the cell and its environment
Also Important in cell communication

Question 3

What are the hallmarks of eukaryotic cells?(7)

Answer 3

Nucleus
Mitochondria
Cytoskeletal structure
Endoplasmic reticular 
Golgi
Lysosomes/peroxisomes/chlorophlasts
Vacuoles

Question 4

What is the role of the nucleus?

Answer 4

Hereditary info stored (DNA)

Question 5

What is the role of the mitochondria?

Answer 5

Produces energy

Question 6

What is the role of the cytoskeletal structure?

Answer 6

Determines cell shape and important in movement of cells and of products within cells, creates network within eukaryotic cell

Question 7

What is the role of the endoplasmic reticulum?

Answer 7

Involved in production and secretion of proteins

Question 8

What is the role of the Golgi?

Answer 8

Transport in cells

Question 9

What is the role of lysosomes/peroxisomes/chloroplasts?

Answer 9

Breakdown of materials

Question 10

What is the role of vacuoles?

Answer 10

Keep structure of cell and store cell sap

Question 11

What is the central dogma of molecular biology?

Answer 11

Flow of information form DNA to RNA (Transcription) {can be reversed} and then RNA to protein (Translation)

Question 12

What is the monomer of fatty acid?

Answer 12

Acetyl coA

Question 13

Why are biomolecules broken down into subunits?

Answer 13

To be used as sources of chemical energy or synthesized to make the chemicals that the cell needs

Question 14

What are nucleic acids synthesised by and degraded by?

Answer 14

Synthesised by polymerases

Degraded by nucleases

Question 15

What are proteins made by and degraded by?

Answer 15

Made by ribosomes

Degraded by proteases

Question 16

Why do some RNAs have catalytic properties?

Answer 16

RNA can interact specifically with other molecules

Question 17

What is primary structure in proteins?

Answer 17

Order of amino acids

3 DNA bases = 1 amino acid

Question 18

What is secondary structure in proteins?

Answer 18

Regular, repeating structures between amino acids close
together in the primary sequence

stabilised by hydrogen bonds between amino acid NH and CO groups

Question 19

What are protein Domains?

Answer 19

Proteins (like myoglobin) fold into a single compact structure separated by flexible regions that are less tightly folded

Question 20

What do protein domains do?

Answer 20

Domains often carry out a specific part of the protein’s
function ( e.g 3 diff domains can carry out 3 diff funtions)

Question 21

What is the tertiary structure in proteins?

Answer 21

Tightly-packed thermodynamically stable 3D structure of the protein (Determined by non-covalent interactions between the side chains)

Question 22

What is the quaternary structure of a protein?

Answer 22

Complex polypeptide structure with two or more subunits

Question 23

How is Protein Targeting (protein moved to membrane/out of cell where it is required by organelle) possible?

Answer 23

Many proteins contain a short signal or localisation sequence showing where they need to go

Question 24

Name some post-translational modifications?(2)

Answer 24

1) Removal of specific parts of the sequence (eg of signal peptides)
2) Addition of small molecules, modulating protein function

Question 25

Name 4 types of addition of molecules in post-translational modifications? (4)

Answer 25

Methylation: addition of -CH3 group(s) Glycosylation: addition of various sugars Ubiquitination: addition of a 76-aa polypeptide Phosphorylation: reversible addition of phosphate (PO3) groups

Question 26

What class of enzymes cause phosphorylation? What is the function of phosphorylation?

Answer 26

Kinases An important way of regulating enzyme function • Phosphorylation of amino acids in or around the active site can change the properties of the region and alter substrate binding

Question 27

What is the Sutton-Boveri theory of chromosome inheritance?(3)

Answer 27

Chromosomes are required for embryonic development. Chromosomes carry Mendel’s “factors” (Sutton used the term gene). Chromosomes are linear structures with genes along them.

Question 28

What were mendels laws of inheritance?(3)

Answer 28

- Segregation - Independent assortment - Dominance

Question 29

How did Griffith show the demonstrate the transforming principle?

Answer 29

Griffith: heat killing of bacteria showing that hereditary material can pass from Smooth to Rough bacteria and change their genotype

Question 30

How did Oswald Avery, Colin MacLeod and Maclyn McCarthy test for molecule responsible for transporting principle?

Answer 30

These scientists systematically destroyed each component of the S strain extract using enzymes that specifically digest each type of molecule (only mouse with DNA destroyed survived)

Question 31

How did Hershey and chase test for what carries bacteriophage genetic material?

Answer 31

Hershey and Chase differentially labelled DNA and protein elements of bacteriophage (using radioactive 32P and 35S respectively); separated the “phage ghosts” from the infected bacteria (Tracking the radioactivity showed that it was the DNA rather than protein that was injected – tracked through several generations)

Question 32

What is DNA made up of? (3)

Answer 32

- Deoxyribose sugar at centre - Sugar Phosphates such as ATP (which is triphosphate) - Base (AGTC)

Question 33

What kind of bond is formed between two nucleotides? | and what is released?

Answer 33

- Phosphodiester bond | - Water molecule

Question 34

If base A is 30% what is T?

Answer 34

70%

Question 35

``` What class of base are A and G? What class of base are T and C? ```

Answer 35

- Purines | - Pyrimidines

Question 36

What are the main features of DNA? (5)

Answer 36

A-T and G-C hydrogen-bonded base pairs Antiparallel strands Right-handed double helix One helical turn every 10.5 bp Major and minor grooves

Question 37

How many H bonds between A and T? | C and G?

Answer 37

- 2 | - 3

Question 38

How many carbon rings in purines? | pyrimidines?

Answer 38

- 2 | - 3

Question 39

What polarity is DNA?

Answer 39

5'-3'

Question 40

What is a chromosome?

Answer 40

A long DNA molecule with part or all of the genetic material of an organism

Question 41

What do chromosomes contain? (2)

Answer 41

Genes and regulatory sequences

Question 42

What do bacteria carry to store genetic information? (2)

Answer 42

Circular chromosome | Plasmid (also circular)

Question 43

How are plasmids passed between genes?

Answer 43

conjugation

Question 44

What do plasmids carry? (2)

Answer 44

DNA AB resistance genes (often smaller than circular genome)

Question 45

What are two differences of eukaryotic chromosomes to prokaryotic?

Answer 45

- linear | - found in nucleus

Question 46

What are telomeres?

Answer 46

Repetitive DNA at the ends of linear chromsomes

Question 47

What do telomeres do? (2)

Answer 47

Can be re-lengthened when DNA is lost from the chromosome end during replication Binds specific proteins that differentiate chromosome ends from broken DNA that needs to be repaired

Question 48

What are roles of DNA-binding proteins? (3)

Answer 48

- Regulate gene expression - Cut DNA at specific sequences - Protect DNA

Question 49

What do DNA-binding proteins have an affinity for?

Answer 49

DNA in general or specific sequences (can be single/double strands DNA or haploid and tetraploid)

Question 50

Describe the shelterin complex as a sequence-specific binding of DNA by proteins?

Answer 50

A number of specialised proteins bind telomere DNA, and each other These form a “cap” on the telomere to: -differentiate it from DNA breaks -promote the formation of a special structure in the DNA called a t-loop -recruit telomerase: the enzyme that adds new telomere sequence -protect from nucleases

Question 51

Describe transcriptional regulators as sequence-specific binding of DNA by proteins?

Answer 51

Proteins that bind regulatory sequences near to the promoters of genes to either stimulate or block transcription. Bend the DNA into a favourable or unfavourable shape

Question 52

Describe histones as binding of DNA by proteins?

Answer 52

Eukaryotic genomes are packaged into chromatin. DNA is wrapped around proteins called histones. Beads on a string structure Not sequence-specific Wrap around all DNA

Question 53

Describe Meselson and Stahl experiments on semi-conservative replication of DNA?

Answer 53

Grow bacteria in media containing 15N: make “heavy” DNA Transfer them to media containing 14N: new DNA will be “light” Separate heavy and light molecules by ultracentrifugation

Question 54

What do the results of Meselson and Stahl's experiments on semi-conservative replication of DNA show?

Answer 54

DNA visualised using UV light Showed DNA as semi-conservative 2 bands after 4 generations

Question 55

What is required for DNA replication? (6)

Answer 55

- Primase - DNA polymerase - DNA ligase - Topoisomerase (Okazaki fragments) - Helicase - Single-stand binding protein

Question 56

What is the mechanism of DNA polymerisation?

Answer 56

- Add nucleotides one at a time, in a 5’-3’ direction - Using template strand to form H-bonds: tells which base to add next - Tens or hundreds of nucleotides per second

Question 57

How are DNA nucleotides added?

Answer 57

- Primase adds short RNA - Primer gives DNA polymerase something to work off - Adds DNA nucleotides onto it - Enzymes can remove primer RNA and DNA polymerase can fill in gaps - Last phosphodiester bond made of ligase - Gap joined up by ligase

Question 58

Describe the structure of DNA replication?

Answer 58

Leading strand: 5’-3’ DNA synthesis points towards the replication fork and can proceed continuously Lagging strand: 5’-3’ DNA synthesis points away from the replication fork and must therefore be discontinuous (primed numerous times) Non-spilt area known as parental strand

Question 59

Where is the primer found on the strand in DNA replication?

Answer 59

On the the 3' end | DNA synthesised away from the primer

Question 60

What are Okazaki fragments?

Answer 60

- The pieces of DNA that are stuck together to make up the lagging strand of replication - Several primers needed

Question 61

What is replication bubble?

Answer 61

- Two replication forks from each origin of replication | - lagging strand has fragments first whereas leading has fragment last

Question 62

What does Topoisomerase do in DNA replication?

Answer 62

Topoisomerase relieves pressure from over-winding around the replication bubble by making and resealing breaks in the DNA

Question 63

What does Helicase do in DNA replication?

Answer 63

Helicase breaks hydrogen bonds between the two DNA strands, separating them

Question 64

What does a single-strand binding protein do in DNA replication?

Answer 64

When bubble opened, singe-strand binding protein binds to single stand of DNA and stops H bonds from reforming

Question 65

What type of DNA replication is on the lagging strand? | leading strand?

Answer 65

- Discontinuous replication - Continuous replication So DNA replication is semi-discontinuous

Question 66

Why is there erosion of genetic material at the ends of linear chromosomes?

Answer 66

Last piece can’t be replicated, 3 prime overhang, terminal gap can’t be filled so in every replication we lose a little bit of the chromosome (only on lagging strand) - Erosion - Lose a few DNA nucleotides at the end of every chromosome

Question 67

How do we stop erosion of genetic material at the ends of linear chromosomes?

Answer 67

Telomeres - Repetitive nature helps it bind to specific chromosomes - Short stretches are lost from telomeres at each round of replication instead - Telomerase can help it replenish the telomeres from RNA template

Question 68

What is RNA made up of?

Answer 68

- nucleotides comprise of a heterocyclic base (adenine, cytosine, guanine or uracil) - a ribose sugar unit - a phosphate group.

Question 69

Describe the stem-loop structures in RNA? (7)

Answer 69

- RNA is single stranded - Nucleotides in RNA base pair with each other - Forms step-loop structures with an irregular non helical structure - Noncanonical base-pair interactions are also observed such as G U - Helicical regions of RNA also have major/minor groove - Major groove more based on base pairs - Sugar units more accessible in minor groove

Question 70

What is canonical base pairing?

Answer 70

- Between AU CG (in RNA) | - Can also be non canonical in RNA

Question 71

Describe the secondary and tertiary structure of RNA?

Answer 71

- Stem-loop structures are secondary structures - Tertiary interactions can involve canonical base-pair interactions between single stranded regions that restrict RNA folding

Question 72

What is the most common tertiary interaction within RNA?

Answer 72

- The A-minor motif - involves adjacent adenosine nucleotides inserted into the minor groove of an RNA stem that interact along the edge of a base-pair within that stem structure.

Question 73

What does RNA Polymerase active site contain?

Answer 73

-A short RNA/DNA heteroduplex Destabilisation of the heteroduplex (due to inherent base-pair instability or recruitment of destabilising proteins) causes release of the RNA polymerase from the DNA and transcription termination

Question 74

Where is transcription initiated?

Answer 74

At the promoter region of a gene, where the RNA polymerase binds

Question 75

Where is transcription stopped?

Answer 75

Terminator region of a gene

Question 76

what is the structure of the core RNAP in bacteria?

Answer 76

RNA polymerase is a pentomeric protein complex containing 2 a subunits, a b and b' subunit, and an w subunit

Question 77

What do different protein subunits in bacterial RNAP do? B a w

Answer 77

B - catalytic a - binds transcription factors w - assembly and stability

Question 78

What factors target bacterial RNA polymerases to the promoter regions of genes?

Answer 78

Sigma factors

Question 79

What do sigma factors do?

Answer 79

- Sigma factor enables the scanning polymerase to bind to the gene promoter - The polymerase adopts an open, active conformation -The double-stranded DNA in the active site is melted to form a transcription bubble. A short RNA primer is formed -Sigma factor is released and the polymerase moves away from the promoter (“promoter clearance”) becomes fully engaged in RNA synthesis.

Question 80

Eukaryotic cells have 3 nuclear RNA Polymerases, what are they and what do they transcribe? (3)

Answer 80

RNAP 1 - rRNA RNAP 2 - mRNA, noncoding RNAs RNAP 3 - tRNA, 5s rRNA

Question 81

Recruitment of eukaryotic RNA polymerase II to gene promoters requires a common set of protein factors known as what?

Answer 81

-General transcription factors

Question 82

How is RNAP 2 assembled at eukaryotic gene promoters by general transcription factors ?

Answer 82

Assembly is initiated by the binding of the protein complex TFIID to an A/T-rich sequence in the promoter region known as the TATA box. Assembly generates a pre-initiation complex (PIC), that is primed for transcriptional activity.

Question 83

What is the difference between transcription/translation transition in bacteria vs eukaryotes?

Answer 83

-The two principal steps in gene expression (transcription and translation) are spatially segregated in humans as they occur in nucleus and ribosomes (responds to transcriptional signals slower but provides additional potential points of regulation) -Transcription and translation are coupled in bacteria so organism can respond very quickly to signals that impact on gene expression at the transcriptional level

Question 84

Eukaryotic mRNA processing comprises three main steps: | 3

Answer 84

- capping of the 5’ end - a non-coded nucleotide is added to the 5' end of the transcript co-transcriptionaly -removal of introns (pre-mRNA splicing) - 3’ end processing (cleavage and polyadenylation) - additional adenylate residues added (polyadenylation), this is coupled to co-transcriptional cleavage of the RNA so transcript is cleaved

Question 85

Describe the “m7G cap” structure of eukaryotic mRNA? (3)

Answer 85

- A guanosine nucleotide is added to the 5’ end of RNA pol II transcripts - The cap is linked to the transcript by a 5’-5’ triphosphate linkage - The cap nucleotide is methylated (“m7G cap”). The first transcribed nucleotide is often modified

Question 86

What is the difference between mRNAs in prokaryotes and eukaryotes?

Answer 86

- In Prokaryotes a single polycistronic mRNA transcript is translated into multiple, functionally related proteins in bacterial cells - In Eukaryotes, mRNAs encode a single polypeptide. The expression of functionally related genes is coordinately regulated, gene families are called regulons

Question 87

Why is the genomic DNA sequence encoding a protein within a mammalian cell is typically much longer than that encoding the same sized protein in a bacterial cell?

Answer 87

The protein coding sequence in eukaryotic cells is usually discontinuous, consisting of short stretches that are "edited" together (the exons) and separated by long regions that are removed (the introns) during the process of pre-mRNA splicing. A long initial transcript is undergoes splicing to generate the contiguous sequence for translation

Question 88

How does pre-mRNA splicing occur accurately?

Answer 88

- Intronic and exonic sequences are distinguished through the recognition of splice site sequences - The 5’ splice site sequence GU and the 3’ splice site sequence AG are highly conserved. Introns also contain a “branchpoint” A - Specific proteins also bind to sequences within exons, allowing their identification

Question 89

Pre-mRNA splicing is carried out by a large complex containing RNA and protein called what?

Answer 89

``` The spliceosome (removes introns and keeps axons) ```

Question 90

What are active splicesomes assembled and disassembled by?

Answer 90

-Smaller RNA/protein complexes called “snurps” (small nuclear RNPs).

Question 91

Describe the catalytic mechanism of splicing? (2)

Answer 91

Splicing involves 2 transesterification reactions: (1) The 2’ hydroxyl group of the branchpoint adenosine attacks the 3’ phosphate of the 5’ exon The 5’-2’ phosphodiester bond gives a looped lariat (2) The generated 3’ hydroxyl group attacks the 5’ phosphate of the 3’ exon, releasing the lariat from the spliced exon

Question 92

What are enzymes with an RNA catalytic subunit known as?

Answer 92

Ribozymes

Question 93

What aligns the reacting chemical groups so that splicing occurs?

Answer 93

The Spliceosome

Question 94

How can reacting chemical groups align without a spliceosome?

Answer 94

This can occur in the complete absence of protein - the RNA itself has catalytic activity. These RNA enzymes are known as ribozymes. - The self-splicing RNA intron needs to fold up is a very specific structure to enable splicing to occur

Question 95

What is the RNA profile of Eukaryotic cells? - Messenger RNA (mRNA) - Ribosomal RNA (rRNA) - Transfer RNA (tRNA)

Answer 95

-Messenger RNA (mRNA) (5%) -Ribosomal RNA (rRNA) (75%) -Transfer RNA (tRNA) (10%) -and some small stable RNAs

Question 96

What is the initiation codon that encodes for methionine?

Answer 96

AUG

Question 97

Describe the 3 reading frames of coding sequences?

Answer 97

Protein-encoding RNA consists of a series of triplet nucleotide codons. There are no nucleotides between codons - the genetic code is said to be nonpunctuated. A sequence has three possible reading frames. Specific triplets define the start codon (initiation codon) and stop codon (termination codon), determining the reading frame.

Question 98

Describe the experiment to determine genetic code in vitro?

Answer 98

Analysis of the genetic code was possible with in vitro translation systems using extracts from E. coli - isolate cell extract - degrade mRNA with RNase - inactivate RNase. - add RNA and amino acids (one is 14C-labelled) - precipitate protein & collect by filter binding. -showed that the triplet UUU encodes phenylalanine.

Question 99

What was the experiment to determine genetic code with ribosome binding?

Answer 99

Filter-binding assays were used to test for complex formation of ribosome/tRNA/codon complexes (ability to facilitate ribosome binding by each charged tRNA)

Question 100

What does genetic code being degenerate mean?

Answer 100

-More than one codon can have the same meaning

Question 101

Describe the genetic code? | -How many codons?

Answer 101

- 61 codons with specific genetic meaning - UAG,UAA,UGA are stop codons - AUG (methionine) - UGG (tryptophan)

Question 102

What is degeneracy of genetic code achieved by?

Answer 102

Isoacceptor tRNAs, that are charged with the same amino acid and recognise different synonymous codons (with the same genetic meaning), and by some tRNAs recognising more than one codon due to "wobble"

Question 103

What is 'wobble' in genetic code?

Answer 103

the ability of the first nucleotide in the tRNA anticodon to base-pair with different nucleotides at the third position in the codon

Question 104

What are the 21st and 22nd amino acids that are incorporated during translation? (2)

Answer 104

- Selenocysteine | - Pyrrolysine

Question 105

How are selenocysteine and pyrrolysine incorporated into protein during translation? (2)

Answer 105

Selanoproteins are synthesised by incorporating the amino acid selenocysteine. UGA codons in some contexts can be decoded by tRNASec. In some archaebacteria, the stop codon UAG has been reassigned to encode the amino acid pyrrolysine.

Question 106

What are tRNAS generated from?

Answer 106

Longer precursor molecules.

Question 107

How are tRNAs are generated from larger precursors?

Answer 107

- The 5' end of mature tRNAs is generated through endonucleolytic cleavage by the ribozyme RNPase. - The 3' end is made by endo- or exonucleases. - All tRNAs have a CCA sequence at the 3' end which can be genomically encoded (in bacteria) or added by the enzyme tRNA nucleotidyltransferase (in eukaryotes). - About 10% of eukaryotic tRNAs contain introns. tRNA splicing is mechanistically different from pre-mRNA splicing.

Question 108

Describe the tRNA structure?

Answer 108

tRNAs have a “cloverleaf” structure. The 5’ and 3’ ends are drawn together. The amino acid is attached to the 3’ hydroxyl group of the 3’ terminal A nucleotide. Specific nucleotides within tRNAs are post-transcriptionally modified. Modification of the 1st position of the anticodon allows “wobble”.

Question 109

How does tRNA fold into an L shape?

Answer 109

tRNAs are folded into a flat "L" shape through coaxial stacking of pairs of helices and through base-pairing between the tips of the D and TYC stem-loops. The aminoacyl 3' end of the tRNA molecule is separated from the anticodon loop

Question 110

How are tRNAs charged

Answer 110

tRNAs are “charged” with the appropriate amino acid by aminoacyl-tRNA synthetases: - A single aminoacyl tRNA synthetase charges all isoacceptor tRNAs. - The reaction requires ATP. - The amino acid is linked to tRNA by an ester linkage between the carboxylic acid group of the amino acid and the 3’ hydroxyl group of the terminal nucleotide.

Question 111

What are the 2 steps of tRNA aminoacylation?

Answer 111

- The amino acid and ATP first react to form an aminoacyl adenylate - The aminoacyl group is then transferred to the tRNA. The formation of the aminoacyl adenylate intermediate allows kinetic proofreading

Question 112

What is kinetic proofreading?

Answer 112

The aminoacyl group of intermediates generated from the correct amino acid are efficiently transferred to the tRNA, while intermediates generated from incorrectly selected amino acids are more readily dissociated than processed.

Question 113

Why does tRNA aminoacylation take place?

Answer 113

Because substrate binding alone does not provide sufficient discrimination between chemically similar amino acids.

Question 114

Explain tRNA identity?

Answer 114

- Aminoacyl-tRNA synthetases recognise all isoacceptor tRNAs and distinguish them from noncognate tRNAs. - tRNA identity is known as the “second genetic code”. - tRNA recognition involves both positive identity and negative identity elements, typically in the anticodon loop and the acceptor stem.

Question 115

Describe the structure of a ribosome?

Answer 115

-Consist of two unequally sized RNP subunits - Each subunit is comprised predominantly of (ribosomal) RNA and ~ 20 (in the small subunit) - 50 (in large subunit) unique proteins (the large subunit contains two copies of two proteins) - Each subunit contains one large rRNA; the large subunit also contains 2-3 small rRNAs.

Question 116

Where does the process of translation occur on the ribosome?

Answer 116

The interface of the two subunits

Question 117

Where does codon/anticodon interaction occur on the ribosome?

Answer 117

Decoding centre on the surface of the small subunit

Question 118

Where does peptide bond formation occur on the ribosome?

Answer 118

The peptidyltransferase centre on the surface of the large subunit

Question 119

Where is the newly synthesised polypeptide chain is extruded through in the ribosome?

Answer 119

The exit tunnel that passes through the large subunit

Question 120

How many tRNA binding sites do ribosomes have?

Answer 120

Ribosomes have three tRNA binding sites: tRNA moves from the A (aminoacyl-tRNA), P (peptidyl-tRNA) and E (exit) sites

Question 121

What is peptide bond formation catalysed by?

Answer 121

RNA

Question 122

Describe how tRNAs bind to ribosomes?

Answer 122

The flat, L-shaped tRNAs are aligned next to each other at the subunit interface such that the anticodon loop is directed towards the small subunit and the aminoacyl acceptor stem is directed towards the large subunit.

Question 123

Describe ribosome synthesis in eukaryotes?

Answer 123

Ribosome synthesis involves transcription of rRNA genes, rRNA processing and modification, and assembly with ribosomal proteins. The process has a high energy burden and requires hundreds of protein and RNAs. Transcription and early processing occurs in the nucleolus. Later steps occurs in the nucleoplasm and cytoplasm – the later ensure that functionally active ribosomes are excluded from the nucleus.

Question 124

Describe the process of peptide bond formation? (peptidyltransfer)

Answer 124

- tRNA and mRNA interaction in decoding center - Molecule with amino acid charged and added to the end on chain, charged tRNA attaches to end of codon, charged amino acid bind to tRNA to add carboxyl group - Peptide bond formed, and ester linkage broken to produce uncharged tRNA in that position - Charged tRNA added initially -Polypeptide chain added from 1 tRNA to other tRNA Transfer of polypeptide chain to different tRNA molecule

Question 125

What are the three ribosomal tRNA binding sites? (3)

Answer 125

- the A (aminoacyl) site - the P (peptidyl) site - the E (exit) site

Question 126

Describe the translation elongation cycle?

Answer 126

After peptide bond formation, the polypeptide chain is extended by one amino acid (at the C-terminal end) and transferred from the tRNA in the P-site to the tRNA in the A-site. The two tRNAs in the P and A sites are then translocated into the E and P sites, respectively. upon binding of an aminoacyl-tRNA to the A-site, the deacylated tRNA dissociates from the E site.

Question 127

Describe how GTPases are used in translation elongation?

Answer 127

aa-tRNA is brought to the ribosome by the elongation factor EF-Tu (EF1A in eukaryotes) Translocation requires another elongation factor, EFG (EF2 in eukaryotes) EF-Tu and EFG are GTPases. (GTP to GDP) 2 GTP molecules are hydrolysed per incorporated amino acid.

Question 128

Where must initiation codon (AUG) be positioned in the tRNA?

Answer 128

The P site

Question 129

what must be targeted to the AUG start codon in the P site?

Answer 129

Methionyl-tRNA

Question 130

What are the two distinct methionyl-tRNAs?

Answer 130

- Elongator tRNAs | - Initiator methionyl-tRNA

Question 131

``` What are Elongation-tRNAs and Initiator methionyl-tRNA bound by? ```

Answer 131

Elongation-tRNA - EF-Tu Initiator methionyl-tRNA - IF2 (both GTPases)

Question 132

What is the purpose of Elongator methionyl-tRNAs ? initiator methionyl-tRNA ?

Answer 132

- Elongator methionyl-tRNAs are used to recognize internal AUG codons - Methionyl-tRNA recognizes the AUG start codon

Question 133

What is the Shine-Dalgarno (SD) sequence within the mRNA is recognized by?

Answer 133

base-pairing with nucleotides at the 3’ end of the 16S rRNA

Question 134

Where does the SD/anti-SD interaction position the initiation codon?

Answer 134

In the ribosomal P-site

Question 135

Describe translation initiation in E. coli?

Answer 135

The initiator methionyl-tRNA/IF2 complex associates with the small subunit (SSU) after positioning of the mRNA. The large subunit (LSU) is then recruited. Hydrolysis of GTP causes release of IF2 and formation of the initiation complex In bacteria, base-pairing between rRNA and the Shine-Delgarno sequence ~ 10 nucleotides upstream of the initiation codon positions the initiation codon correctly in the P site. This mechanism allows recognition of multiple initiation codons within a polycistronic mRNA

Question 136

Describe translation initiation in eukaryotes?

Answer 136

Met-tRNA is bound by eukaryotic initiation factor 2 (eIF2) The Met-tRNA/eIF2 complex binds to the SSU(small sub unit) and is recruited to the 5’ end of the mRNA Interaction of the Met-tRNA/eIF2/SSU complex with the mRNA is dependent upon the cytoplasmic cap binding complex The Met-tRNA/eIF2/SSU/CBC complex scans along the mRNA, using the helicase activity of CBC, until it finds an AUG codon within an appropriate context: the Kozak sequence. (has to be within particular sequence) GTP hydrolysis by eIF2 leads to its release, recruitment of the LSU and formation of the translation initiation complex

Question 137

What are termination codons recognised by?

Answer 137

Termination factors | also known as release factors

Question 138

What does binding of termination factor trigger?

Answer 138

peptide hydrolysis

Question 139

What are the termination factors?

Answer 139

Release factors RF1 or RF2 (eRF1 in eukaryotes)

Question 140

What allows for release release factors to be released?

Answer 140

RF3 for RF1 and RF2 | eRF3 in eukaryotes

Question 141

What dissociates the ribosomal subunits and mRNA after termination?

Answer 141

A set of factors including EF-G

Question 142

Why is Gene Expression Regulated?

Answer 142

Gene regulation ensures that the appropriate genes are expressed at the proper times. Gene regulation can also help an organism respond to its environment.

Question 143

What does it meant to say some genes are constitutively expressed?

Answer 143

Constitutively expressed (not upregulated or downregulated/constant level). The genes are referred to as house-keeping genes because the products are required for essential ongoing functions.

Question 144

What are the two types of mutations that affect regulating gene expression? (2)

Answer 144

cis-acting | trans-acting

Question 145

What do cis and trans mutations identify?

Answer 145

cis - DNA/RNA sequences that affect gene regulation trans - (protein, or potentially RNA) factors that regulate the expression of a target gene.

Question 146

What is the purpose of transcriptional regulation?

Answer 146

Transcriptional regulation is the primary level of control for most genes (first step in pathway). Transcriptional regulation limits wasteful production of unrequired biomolecules

Question 147

What do trans-acting factors do?

Answer 147

Trans-acting factors can act to up- or downregulate expression

Question 148

What factors cause activation?

Answer 148

trans-acting activators

Question 149

What factors cause down- regulation?

Answer 149

trans-acting repressors

Question 150

What do activators do?

Answer 150

- Activators promote expression at weak promoters | - Activators interact with the a-subunit of RNA polymerase and promote DNA binding

Question 151

Explain the promoter region of E. coli genes?

Answer 151

The consensus sequence is the sequence consisting of the nucleotide found at the highest frequency across all genes at each position. Promoter sequences closely matching the consensus sequence interact strongly with sigma factor and allow efficient transcription. Weak promoters have sequences that diverge from the consensus sequence, interact poorly with sigma-70 and show low transcriptional activity

Question 152

How can enzyme-encoding genes be upregulated?

Answer 152

Enzyme-encoding genes can be upregulated in the availability of substrate. The molecule mediating the upregulation is called an inducer. Genes under positive or negative control can be inducible.

Question 153

How can enzyme-encoding genes be downregulated?

Answer 153

Enzyme-encoding genes can be downregulated in the presence of product. The active molecule is called a corepressor. Genes under positive or negative control can be repressible.

Question 154

How can gene expression can be regulated at the level of RNA processing?

Answer 154

Pre-mRNA splicing can occur in different patterns. This can lead to the production of two distinct proteins. Alternatively, there might be a productive and nonproductive pathway. The pre-mRNA can be degraded, blocking expression.

Question 155

What is an alternative of splicing?

Answer 155

Mutual exon exclusion. Exon inclusion or exclusion reflects activator or repressor proteins binding to splice site enhancers or splice site silencer sequences close to splice sites.

Question 156

How can gene expression be regulated at the level of translation?

Answer 156

Translation regulation typically impacts on initiation. Translation of prokaryotic mRNA requires recognition of the Shine-Delgarno sequence. (1) Translation repressors can bind to the mRNA such that the Shine-Delgarno sequence is no longer available for interaction with the small ribosomal subunit. (2) Small molecules can potentially impact on RNA secondary structure to block the Shine-Delgarno sequence. (3) Some mRNAs have structures that can be resolved at increased temperature, providing a temperature-dependent expression pattern.

Question 157

Explain auto regulation of gene expression?

Answer 157

Some genes encoding RNA-binding proteins autoregulate their own expression. Some ribosomal proteins in E. coli bind to their mRNA, blocking the Shine-Delgarno sequence. This is an example of a negative feedback loop, where expression of the ribosomal protein is limited when insufficient rRNA is available for subunit assembly. The mRNA sequence adopts a structure similar to the rRNA binding site of the ribosomal protein.

Question 158

How can protein function be regulated by post-translational modifications?

Answer 158

Many proteins are processed or post-translationally modified. Phosphorylation of serine, threonine or tyrosine residues is the most common form of post-translational modification. Phosphorylation can affect protein conformation or its ability to interact with other proteins. Phosphorylation is readily reversible. Protein dephosphorylation generates free phosphate.

Question 159

What three structural genes does the lac operon code for that are associated with lactose metabolism? (3)

Answer 159

- lacZ - lacY - lacA

Question 160

When is the lac operon expressed?

Answer 160

Expressed only when the cells need to metabolise lactose

Question 161

What is expression of lac operon controlled by?

Answer 161

-lacI | I is an i

Question 162

What encodes the lac repressor?

Answer 162

-lacI | I is an i

Question 163

What does the lac repressor do?

Answer 163

Binds to lacO operator sequences and blocks RNAP activity

Question 164

How is allolactose generated to induce expression of lac operon?

Answer 164

Lactose (milk sugar) is a disaccharide that is hydrolysed by b-galactosidase to glucose and galactose monosaccharides. Galactose can then be phosphorylated and converted to glucose phosphate. Expression of the lac operon is induced by allolactose, a lactose isomer with a 1,6 b-galactosidic linkage. Allolactose is thought to be generated by b-galactosidase in a side reaction and can be metabolised.

Question 165

What is the lacI repressor known as?

Answer 165

A homotetramer

Question 166

What are the three domains of the lacI repressor? (3)

Answer 166

- A tetramerisation domain - A core domain that binds the inducer - A head domain that binds the operator sequence

Question 167

What happens to lac operon when bound to inducer?

Answer 167

The repressor alters shape and cannot bind DNA - it binds DNA and inducer in a mutually exclusive manner. This is an example of an allosteric interaction (change in protein structure, prevents it from binding to another molecule).

Question 168

Why is lac operon expression inhibited in the absence of lactose?

Answer 168

-The repressor is expressed constitutively

Question 169

What happens when the lacI repressor binds to the operator sequences?

Answer 169

The DNA binding sites of each subunit are aligned. This is thought to cause the DNA to twist.

Question 170

What does binding of lacI repressor to operator sites do in regards to RNAP

Answer 170

The RNAP is primed to transcribe lac mRNA immediately upon binding of inducer to the repressor.

Question 171

What is the additional level of control in lac operon expression known as?

Answer 171

Catabolite repression

Question 172

Explain lac operon regulation by catabolite repression

Answer 172

In the presence of glucose, expression of genes required for the metabolism of other sugars including lactose is repressed. When limiting glucose and lactose are both present in the medium, lactose is only metabolized after glucose is exhausted. This causes a diauxic growth curve. A catabolite of glucose represses expression of the lac operon. Expression of the lac operon requires both the presence of lactose and the absence of glucose.

Question 173

Which mutations in genes block expression of the lac operon? (2)

Answer 173

- adenylate cyclase | - the catabolite activator protein (CAP)

Question 174

What does adenylate cyclase do?

Answer 174

Adenylate cyclase converts ATP to cyclic AMP and is inhibited in the presence of glucose.

Question 175

What does CAP do in regards to cAMP?

Answer 175

cAMP binds to CAP. The cAMP/CAP complex binds to the CAP site and is required for RNAP activity.

Question 176

When is the lac operon expressed?

Answer 176

In the absence of glucose and presence of lactose

Question 177

Why is eIF2 important in translation initiation?

Answer 177

Eukaryotic initiation factor 2 (eIF2) binds to the charged initiator tRNA Met-tRNA and brings it to the small ribosomal subunit. Once the initiation codon has been localized, eIF2 hydrolyses GTP. This causes a conformational change in eIF2, releasing it from the ribosome bound to GDP.

Question 178

Why is eIF2 a GTPase molecular switch?

Answer 178

eIF2 has weak GTPase activity and is dependent upon proteins that are its GTPase activating protein (GAP) and a guanine exchange factor (GEF) to hydrolyse GTP and be reactivated after GTP hydrolysis, respectively. The protein is a heterotrimer.

Question 179

Why does eIF2 have weak GTPase activity?

Answer 179

The y subunit has GTPase activity and binds to the initiator tRNA, while the a subunit has a regulatory function.

Question 180

Why can eIF2 activity be rate-limiting for protein synthesis?

Answer 180

Cells downregulate global translation in response to various stress signals by depleting the active pool of eIF2. This is known as the integrated stress response in mammalian cells Protein kinases phosphorylate Ser-51 in the a subunit in response to a wide range of stress signals.

Question 181

What does phosphorylation of eIF2 do?

Answer 181

Phosphorylation of eIF2 does not affect GTP hydrolysis, interaction with eIF2B or GDP release. However, eIF2-Pi binds tightly to eIF2B. There is about 10 times more eIF2 than eIF2B in the cell. Phosphorylation of eIF2 converts it from a substrate of eIF2B to an inhibitor.

Question 182

vector + gene=

Answer 182

Recombiant DNA

Question 183

What enzyme cuts up DNA

Answer 183

Restriction enzymes

Question 184

What do the 4 types of restriction enzyme do? (4)

Answer 184

Types I and III cleave DNA at sites away from recognition sequence. Type IV cleave modified DNA. Type II cut DNA at a defined position, either within or near to their recognition site.

Question 185

Which type of restriction enzyme is widely used in cloning?

Answer 185

Type 2 | Can cut DNA at defined position

Question 186

Describe type 2 restriction enzymes?

Answer 186

Type II REs are homodimers – two identical polypeptides. Sequence-specific – single nucleotide change eliminates activity, short so between for and 8 base pairs. 6bp cutters most commonly used in molecular biology. Palindromic – reads same 5’-3’ on each strand. Ends can be blunt (SmaI) or overhanging (HindIII) – also called “sticky” ends Next slide: how a restriction enzyme works

Question 187

How does a restriction enzyme work?

Answer 187

Initial binding is non-specific: looser, catalytic site not involved (no specific cutting) Enzyme then moves along DNA: it can “slide” for short distances but can also jump or hop over longer distances if it doesn’t encounter a specific site Recognition of a specific site -> conformational changes (enzyme and DNA). Exact mechanism not yet known Ends have 5’-phosphate and 3’-OH groups

Question 188

Some DNA ends are overhanging, what do these pair with?

Answer 188

Other compatible overhanging ends

Question 189

What sticks DNA together?

Answer 189

DNA ligase

Question 190

Describe the ligation reaction (sticking two DNAs together)

Answer 190

1) First the ends have to interact with each other (hydrogen bonding) Inefficient – lower temperatures help (slower molecular movement; stabilises H-bonds for sticky ends) 2) If step 1 happens for long enough, DNA Ligase catalyses the formation of a phosphodiester bond (between phosphate and OH groups) Better at higher temperatures (25 C optimal)! Compromise – 1h at 16 C / overnight at 4 C

Question 191

Describe the DNA ligase mechanism?

Answer 191

AMP is transferred to a lysine residue in the enzyme’s active site (from ATP – the cofactor). AMP is then transferred to the 5′-phosphate. The AMP-phosphate bond is attacked by the 3′-OH, forming the covalent bond and releasing AMP. ATP is required to replace the AMP used in the reaction (i.e. is a cofactor)

Question 192

What are potential problems in DNA cloning? (2) | And their solutions?

Answer 192

1) The vector has complementary ends – it might ligate to itself (very likely in fact – closer together), could join itself and modify ends Modify vector ends – phosphatase treatment removes 5’ phosphate – no phosphodiester bond can be formed 2) Gene may insert in wrong orientation Use more than one enzyme (also solves first problem/no longer compatible end) for each end

Question 193

How do you add a 5' phosphate?

Answer 193

(T4 polynucleotide Kinase) | Required if there isn't a phosphate group

Question 194

How do you remove a 5' phosphate group

Answer 194

(Calf Intestinal Phosphate) CIP “Sticky” ends can still base pair via hydrogen bonding but ligation can no longer occur Can be used to prevent self-ligation of your vector

Question 195

Why is a DNA overhang removed? (2)

Answer 195

Blunt end cloning might be necessary Destroy restriction enzyme sites

Question 196

How do you get rid of overhang? (3)

Answer 196

# Fill with complementary bases Use enzyme to chop off 5’ overhang Use enzyme to chop off 3’ overhang

Question 197

What are the essential features of all vectors? (3)

Answer 197

Origin of replication (Independent replication inside the host) Selectable marker (Survival of host cells that are carrying your plasmid) Multiple Cloning Site Restriction enzyme sites Unique sites Where to clone your gene

Question 198

What is the most common vector?

Answer 198

Plasmid is most common – circle of DNA that exists outside of the chromosome in bacteria. Carries genetic information.

Question 199

How do you recombine DNA into cell in molecular cloning?

Answer 199

Electroporation Brief pulse of high-voltage thought to induce transient pores in cell membranes Chemical Transformation Chemically treated E. coli Add a heat-shock Causes cell membrane changes that allow uptake of DNA

Question 200

How do you select bacteria with plasmid?

Answer 200

Use selectable marker on your vector

Question 201

What does PCR stand for?

Answer 201

Polymerase Chain Reaction

Question 202

What do we need for PCR? (6)

Answer 202

Template – DNA of some sort DNA polymerase – an enzyme that copies DNA Primers – DNA polymerases need a free 3’-OH to start. (Therefore, we need to know a bit about our sequence) Deoxyribonucleotide triphosphates (dNTPs) – DNA bases to make the new DNA strand Appropriate buffer – MgCl2 Appropriate temperature – each PCR cycle consists of stages. We use a thermocycler

Question 203

What happens during PCR?

Answer 203

Imagine we start with one molecule of DNA After one cycle, we have doubled this. Each cycle -> doubling of DNA molecules = exponential After each cycle – molecules of DNA = multiply by 2 to the power of the number of cycles

Question 204

What are the three stages of a PCR cycle? (3)

Answer 204

1) Denaturation 95°C Double-stranded DNA dissociates into single-stranded DNA. 2) Primer Annealing 55-65°C Primers bind to complementary sequence on ssDNA. Primer binding is antiparallel. 3) Primer Extension 68-72°C DNA polymerase synthesises new strands of DNA from the 3’ end of the primers.

Question 205

How many times is PCR cycle repeated?

Answer 205

30 times, newly synthesised DNA is the template for the subsequent cycle

Question 206

What DNA polymerases are used in PCR? (2)

Answer 206

Taq Pfu

Question 207

What attributes are important in DNA polymerases for PCR? (4)

Answer 207

Thermostability is important Extension rate – how fast it can replicate a template Processivity – how often it falls off and has to re-associate Proof-reading and fidelity – contribute to accuracy. Pfu introduces fewer mutations into its PCR products

Question 208

What are important features of PCR primers? (3)

Answer 208

Specific to your template Come in pairs: must bind opposite strands in opposite orientation Around 20 bp in size (17 is a minimum)

Question 209

What does the melting temperature of primers mean?

Answer 209

Temperature at which the primer will dissociate from the DNA template Primer Tm determines what annealing temperature (Ta) to use in your PCR cycle – should be about 5°C lower than the Tm

Question 210

What happens to primer binding if annealing temperature is too low? too high?

Answer 210

Ta is too low: primers may bind non-specifically to other DNA sequences Ta is too high: primers may not bind efficiently (or at all), reducing product yield

Question 211

What are the issues with directly cloning your PCR product? (4)

Answer 211

Taq adds a 3’ A overhang to its products -Can remove it -[Clever vectors that use the A overhang – TA cloning] PCR products have no 5’ phosphate - Fine if your vector has a 5’ phosphate - But then your vector will self-ligate! Blunt-ended cloning is not very efficient Blunt-ended cloning is not directional

Question 212

What is the process of Reverse Transcription PCR (RT-PCR)

Answer 212

RNA is reverse transcribed into DNA (called complementary DNA or cDNA) PCR is used to amplify a specific cDNA sequence

Question 213

What are the uses of Reverse Transcription PCR (RT-PCR)

Answer 213

Molecular cloning – sometimes we want a cDNA sequence, rather than the whole genomic sequence (protein expression). Last time, talked about our template for PCR – cDNA is often a template, rather than genomic DNA. RNA expression: Is a gene being expressed? How much mRNA is being produced? Analysis of the different isoforms of mRNA.

Question 214

Describe cDNA synthesis in RT-PCR?

Answer 214

First Strand Synthesis 1) Reverse transcriptase synthesis the first strand of cDNA 2) Poly(dT) primers bind poly(A) tail of mRNA 3) RNA is removed Second Strand Synthesis 1) Synthesised by the Klenow fragment of DNA polymerase I 2) The hairpin formed by RT acts as a primer 3) The ssDNA loop can be digested by a nuclease

Question 215

What is qPCR?

Answer 215

Quantitative PCR

Question 216

What does qPCR do?

Answer 216

qPCR looks at exponential phase (i.e. in real time) – when amount of PCR product crossed a threshold level (background)

Question 217

How to measure your qPCR product?

Answer 217

1)Fluorescent dye SYBR Green Fluoresces when it binds dsDNA (double-stranded DNA) Fluorescence is proportional to amount of dsDNA Not sequence specific 2) Fluorescent probes Sequence specific Can multiplex (several targets in 1 reaction) Fluoresces when displaced from template

Question 218

Difference between RT-PCR (reverse transcriptase PCR) and qPCR?

Answer 218

qPCR is often referred to as Real-Time PCR, causing some confusion with RT-PCR Some overlap: qPCR is frequently RT-PCR, as it’s used for analysing mRNA levels. But it doesn’t have to be – possible to perform qPCR on templates other than RNA Pathogen detection And we can perform RT-PCR that is not quantitative Molecular cloning

Question 219

What is the purpose of DNA sequencing?

Answer 219

Make sure you have cloned your target sequence Check there are no PCR-induced mutations Sequence a gene (WT and mutant) Check success of mutagenesis (Sequencing is a specialised form of DNA synthesis)

Question 220

What do you need to know for DNA sequencing? (2)

Answer 220

What is the last nucleotide How long is the DNA molecule

Question 221

How does DNA sequencing work?

Answer 221

Once we know last nucleotide and length of DNA Then we can work out what nucleotide is at a specific position So, if we stop DNA synthesis specifically at the nucleotide “A”, and we get fragments of size 1, 5, 10 and 12…

Question 222

How do you find out which base is last and which DNA molecule is longest?

Answer 222

How do we know which base is last? Use different ddNTPs: ddATP, ddTTP, ddGTP, ddCTP Originally, four separate reactions were set up…each with a different ddNTP Products resolved by electrophoresis How do we know how long the DNA molecule is? Gel electrophoresis – discriminate between DNA molecules by size

Question 223

In DNA sequencing, if sequencing terminates at the first A (e.g.) how will we ever know the rest of the sequence?

Answer 223

We use both ddNTP + “normal” dNTPs (usually an excess of these). Millions of products in our sequencing reaction. Each will incorporate the ddNTP in a different place

Question 224

What are the components of DNA sequencing reaction? (6)

Answer 224

Template DNA – plasmid / PCR product Primer – do we need to know our sequence? - We usually use a primer that binds the vector - Can design more primers once we know the sequence A DNA polymerase (T7 DNA polymerase) Deoxynucleotides (dNTPs): dATP, dCTP, dGTP, dTTP Dideoxynucleotides (ddNTPs) – fluorescently labelled ddATP, ddCTP, ddGTP, ddTTP Buffer (including Magnesium)

Question 225

What are DNA sequencing products labelled with?

Answer 225

Fluorescent tag The ddNTPs have different coloured tags – they can all go in one reaction

Question 226

What is gene expression?

Answer 226

The production of a functional RNA or protein from the genetic information encoded by the genes. Differential expression of genes results in different cells, tissues and organs

Question 227

How do Hybridisation-based techniques of gene expression work?

Answer 227

Based on properties of DNA/RNA – complementary sequences will hybridise to each other – DNA/DNA or DNA/RNA If you have a sequence that you are interested in (RNA/cDNA/DNA), you can use that sequence to make a “probe” Probe = DNA sequence that is complementary to the RNA sequence you want to analyse – labelled in some way that we can analyse (various ways to do this)

Question 228

What are the hybridisation-based techniques of gene expression?

Answer 228

RNA expression: - Northern Blot - Microarray RNA localisation -Fluorescence In situ hybridisation

Question 229

Explain the Northern blot in RNA expression?

Answer 229

1) RNA is separated by electrophoresis – will see major bands of rRNA 2) RNAs are transferred to a membrane 3) Probe – complementary to your chosen sequence – labelled (usually radioactive for detection in phosphorimager) - Can tell you about abundance, size, isoforms, expression in different tissues/cell types - Limited to a single gene each time

Question 230

Explain the use of microarrays in RNA expression?

Answer 230

- Oligonucleotides are attached to a spot on a chip - Each spot has a different oligonucleotide, corresponding to a specific gene - RNA is prepared from a source and fluorescently-labelled cDNA is made from the RNA - Fluorescent cDNA is applied to the chip and allowed to hybridise Lot of mRNA -> lot of cDNA ->lot of fluorescence Not much mRNA -> not much cDNA -> low fluorescence

Question 231

What is the role of mircoarrays?

Answer 231

To measure relative mRNA levels Not easy to quantify absolute values of mRNA levels Better for assaying RELATIVE levels (a bit like qPCR) Different fluorescent tags for cDNA from different sources

Question 232

How do we compare levels of transcription with mircoarrays?

Answer 232

cDNA levels are a substitute for RNA levels Black: No cDNA from either source Yellow: Equal cDNA from both sources Green: More cDNA from tumour cells Red: more cDNA from normal cells

Question 233

Explain RNA localisation in gene expression analysis?(Fluorescent In situ hybridisation)

Answer 233

Fluorescent In situ hybridisation = FISH for short Principle of hybridisation is similar to a Northern blot Probe is often labelled with a fluorescent marker and visualised using microscopy Can reveal RNA localisation within a chromosome, a cell, an organ, the whole organism Correct RNA localisation can be vital for correct development

Question 234

How do protein detection methods work in regards to antibodies?

Answer 234

A lot of protein detection methods use antibodies Recognise a specific protein Secondary antibody recognises the primary antibody Usually conjugated to a molecule that allows detection (specific for application)

Question 235

Describe western blotting in protein expression and detection?

Answer 235

1) Proteins are separated by electrophoresis (based on size) 2) Then transferred to a membrane 3) Detection is using a protein-specific antibody and a labelled secondary antibody Last step – recognition uses primary and secondary antibodies. Secondary usually conjugated to an enzyme that produces light Can tell us about protein levels; isoforms (if they’re different sizes); sometimes PTMs, expression patterns in different cells/tissues

Question 236

Describe Immunofluorescence in protein expression and detection?

Answer 236

Not live imaging – a snapshot in time (cells usually need to be fixed and prepared in quite harsh ways) Secondary antibody is usually conjugated to a fluorescent molecule – imaged using microscopy Use different fluorophores – can see more than one molecule at a time Related technique (IHC) – often use enzymatic secondary antibodies

Question 237

What is the use of Fusion proteins for protein localisation?

Answer 237

The fusion proteins is easy to visualise Fuse the protein-coding regions together – visualise the reporter to assess localisation Fluorescent proteins are commonly used – visualised by microscopy

Question 238

What are the properties of reporter genes?

Answer 238

Easy to visualise (like GFP) Or easy to assay (luciferase and β-galactosidase) Fusion proteins are a type of reporter gene

Question 239

What are the tools used for analysing molecular interactions? Protein-protein interactions? (2) Protein-DNA interactions?

Answer 239

Protein-protein interactions: - Pull-down assay - Yeast two-hybrid Protein-DNA interactions: -Chromatin Immunoprecipitation

Question 240

Describe pull-down assay as a tool to analyse protein-protein interactions?

Answer 240

Uses a fusion protein that has affinity for a specific ligand Fuse to the protein-coding sequence of your favourite gene Immobilise ligand on a surface (usually a little bead) Fusion protein will bind – and will bring your favourite protein Analyse what is also bound – these will be interacting proteins A way of analysing protein interactions in vitro

Question 241

How can new partner be identified in pull-down assay when analysing protein interactions?

Answer 241

New partners can be identified by Western blotting or mass spectrometry

Question 242

What does elution mean?

Answer 242

A term referring to removing bound proteins from the affinity column

Question 243

Describe yeast two-hybrid as a tool to analyse protein-protein interactions?

Answer 243

Fusion proteins again… The “bait” is fused to a DNA-binding domain The “prey” is fused to a transcription activation domain Both fusion products are expressed inside a yeast cell, along with… A reporter gene with a promoter that can be bound by the DNA-binding domain If bait and prey interact, this will bring the transcription activation domain to the promoter and induce expression of the reporter gene Report gene is often β-galactosidase – easy to assay as it produces a blue colour Lot of transcription factors are “modular” – DNA-binding and transcription activation can be separated

Question 244

Describe Chromatin immunoprecipitation(ChIP) as a tool to analyse protein-DNA interactions?

Answer 244

Have to cross-link DNA-protein first (or the interaction will not survive harsh purification conditionssing ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA

Question 245

What is the use of Chromatin Immunoprecipitation (ChIP)

Answer 245

Used to study interaction of proteins with DNA in a living cell Use an antibody to purify your protein of interest (could also use a fusion protein too) Assay which DNA molecules are associated with your protein

Question 246

What does a plasma membrane do?

Answer 246

Provides cell boundary and prevents movement of materials in to and out of the cell

Question 247

What do organelle membranes do?

Answer 247

Divide cytoplasm into compartments

Question 248

What are the fundamental properties of membranes?

Answer 248

Barrier Flexible, self-repairing, continuous Selectively permeable - only certain molecules can pass into and out of cells

Question 249

What is the composition of membranes made up of?

Answer 249

Lipids Proteins Carbohydrates

Question 250

What are the ways phospholipids move about in bilayer?

Answer 250

Lateral diffusion- head moves left to right Flexion - tail moves left to right Rotation - phospholipid rotates Flip-flop - whole membrane flips over (requires energy)

Question 251

How does composition of lipid bilayer in terms of bonds affect fluidity?

Answer 251

More double bonds between C atoms, less acyl chains, less tightly packed molecules, greater fluidity

Question 252

What are the properties of phospholipids?

Answer 252

Amphipathic - Polar head is hydrophilic, fatty acid tail is hydrophobic

Question 253

What is the difference in double bonds in membrane between warm-blooded animals and cold-blooded animals/plants?

Answer 253

Less double bonds in Cold-blooded animals/plants

Question 254

What is the structure of cholesterol?

Answer 254

Polar head group Rigid steroid ring structure Nonpolar hydrocarbon tail

Question 255

What does cholesterol do in the membrane?

Answer 255

Makes membrane less permeable Cholesterol goes between two phospholipids, when joined with steroid ring makes membrane less permiable

Question 256

What do membranes form in aqueous solutions?

Answer 256

Micelles (drop) | Bi-layer

Question 257

How to integral proteins insert into membrane?

Answer 257

Directly insert | in the membrane by a hydrophobic domain

Question 258

What do peripheral membrane proteins do?

Answer 258

1) associate with integral membrane proteins or directly bind lipids 2) covalently bind lipids which insert into the membrane

Question 259

What modification do small G proteins have?

Answer 259

Have a fatty acid modification which allows them to cycle on and off membranes: Active on membranes, inactive in the cytosol

Question 260

Explain the formation of rafts in membranes?

Answer 260

Cholesterol and sphingolipids can form microdomains called rafts The membrane is slightly thicker in the raft microdomain

Question 260

Explain the formation of rafts in membranes?

Answer 260

Cholesterol and sphingolipids can form microdomains called rafts The membrane is slightly thicker in the raft microdomain

Question 261

Why isn't there movement between apical (surface) and basolateral (base) membranes

Answer 261

Tight junctions prevent movement between | apical and basolateral membranes

Question 262

What is the plasma membrane linked to?

Answer 262

The cytoskeleton

Question 263

What are key features of biological membranes? (3)

Answer 263

Membranes are asymmetric Proteins always have the same orientation in the membrane The lipid composition of each of the two halves of the bilayer is different

Question 264

What are the blood types? (4)

Answer 264

A B O (is the universal donor) AB (is the universal acceptor)

Question 265

When is membrane asymmetry important?

Answer 265

COAGULATION (clot formation) phosphatidylserine on platelets and other cell membranes provides the nucleation site for the coagulation cascade. CELL RECOGNITION AND CLEARANCE The macrophage plasma membrane contains receptors, which recognise aminophospholipids – phosphatidylserine or phosphatidylethanolamine which are transferred to the outer leaflet of the plasma membrane of apoptotic cells

Question 266

How do membranes transport proteins of small molecules? (3)

Answer 266

Active transport Electrochemical gradients Carriers and channels

Question 267

What kind of molecules are more soluble?

Answer 267

Small non-polar molecules

Question 268

What kind of proteins are membrane transport proteins?

Answer 268

All are multi-pass integral membrane proteins

Question 269

Give an example of passive transport (down conc grad) in membranes? (2)

Answer 269

Simple diffusion | Channel-mediated diffusion

Question 270

Give an example of active transport(against conc grad) in cell membranes?

Answer 270

Carrier protein diffusion

Question 271

How is transport across membrane affected by electrochemical gradients?

Answer 271

More transport when moving toward more negative side

Question 272

What gives you an electrochemical gradient?

Answer 272

Established by ionic concentration differences on either side of the membrane – produced through the action of ion channels and carriers/pumps Drive transport processes, convey electric signals in nerves, make ATP in mitochondria, chloroplasts and bacterial membranes.

Question 273

Why do channel proteins transport faster than carrier proteins?

Answer 273

Don't have to bind to molecule

Question 274

How can ion channels be regulated by being open/closed? (4)

Answer 274

Can be regulated by : Voltage Binding of ligands extracellularly Binding of ligands intracellularly Mechanically gated

Question 275

What can active transport be mediated by? (3)

Answer 275

3 ways to transport solute against gradient: 1) Co-transport, moved by binding and transport of another protein 2) ATP driven pump using energy from phosphorylation 3) Use of light to move solute against gradient

Question 276

What are the three types of carrier-mediated transport? (3)

Answer 276

Uniport - one molecule transported across Symport - coupled transport of two molecules in the same direction Antiport - coupled transport of two molecules in the opposite direction

Question 277

What is mammalian plasma membrane transport driven by?

Answer 277

Na+ gradients

Question 278

What is bacteria, yeast, intracellular membranes | transport driven by?

Answer 278

transport driven by H+ gradients

Question 279

What three carriers does the transport of glucose across epithelial cells involve? (3)

Answer 279

Glucose/sodium ion symporter at the apical surface Sodium/Potassium pump at the basal surface Glucose carrier at the basal surface

Question 280

Describe the transcellular transport of glucose?

Answer 280

Glucose co-transported into lateral domain Glucose co-transported again into extracellular fluid Sodium/Potassium pump moves sodium out and potassium in the lateral domain

Question 280

Describe the transcellular transport of glucose?

Answer 280

Glucose co-transported into lateral domain Glucose co-transported again into extracellular fluid Sodium/Potassium pump moves sodium out and potassium in the lateral domain

Question 281

What are the two hypothesises for the evolutionary origin of the nucleus?

Answer 281

Hypothesis 1 - Invagination of membrane around DNA - Formation of primitive nucleus Hypothesis II - Engulfment and mutual independence to form organelles is knows as endosymbiosis - Strong evidence to support this occurred with mitochondria - May have occurred in nucleus as well

Question 282

How is DNA packaged in the nucleus?

Answer 282

DNA in the nucleus is packaged into chromosomes DNA is wrapped around histones for effective packaging In non-dividing cells, DNA is loosely packed forming a tangle of strands – chromatin Just before cell division, packaging tightens up and chromosomes become visible

Question 283

Describe chromatin organisation in the nucleus?

Answer 283

Heterochromatin: Dense staining of interphase DNA Euchromatin: Less-dense staining interphase DNA Nucleolus: Highly dense staining of RNA

Question 284

Describe chromosome placement in the nucleus?

Answer 284

Chromosomes occupy specific territories within the nucleus, which may be identified by chromosomal painting territories are inherited but can change following differentiation or disease

Question 285

What happens to the location of a gene in regards to transcriptional status?

Answer 285

It changes

Question 286

What are the sub-nuclear organelles in inter-chromatin space of the nucleus? (4) and what do they do?

Answer 286

Nucleolus: ribosome synthesis Speckles: pre-mRNA processing Cajal bodies: splicing PML bodies: storage depot (Sub-nuclear organelles can be dynamic and move in non-random ways in an ATP-dependent manner)

Question 287

Describe the nucleolus of the nucleus?

Answer 287

- It is not membrane bound - Site for processing ribosomal RNA to produce ribosomes -It is a collection of macromolecules including: rRNA genes, precursor rRNA, mature rRNA, rRNA processing enzymes, snoRNPs, ribosomal protein subunits, partly assembled ribosomes The nucleolus also processes other types of RNA mRNA – messenger tRNA – transfer RNA

Question 288

Describe the nuclear envelope of the nucleus?

Answer 288

- Nuclear envelope is a double unit membrane perforated with pores and supported by a fibrous meshwork called the lamina - The lamina is responsible, in part, for ensuring the asymmetric nature of the double unit membrane

Question 289

What is the role of the lamina in the nuclear envelope?

Answer 289

- The lamina is responsible, in part, for ensuring the asymmetric nature of the double unit membrane - Lamina plays a global role in gene regulation as well as structural rigidity

Question 290

Describe nuclear access in the nucleus?

Answer 290

Pore on the nuclear envelope Nuclear access is controlled by the pore in a size-dependent manner The signal for entry is a specific peptide sequence

Question 291

How is the ER involved in lipid asymmetry?

Answer 291

-Lot of phospholipid synthesis occurs at the ER membrane -Newly made lipids incorporated into cytosol -Enzyme scramblase mixes up newly formed lipids and places them on both sides of the bilayer This messes up the symmetry

Question 292

How is the Plasma membrane involved in lipid asymmetry?

Answer 292

- This is done by the enzyme flippase which ensures membrane asymmetry is maintained - Asymmetry can occur by formation of new lipids and also when vesicles fuse with the membrane and deliver its lipids

Question 293

What is flippase also known as?

Answer 293

aminophospholipid translocase

Question 294

What is the role of flipase (aminophospholipid translocase)?

Answer 294

Role is to ensure that any phosphatidylserine delivered to the outer leaflet is retrieved back to the inner leaflet Phosphatidylserine needs to be ready to act as a signal for apoptotic clearance or coagulation

Question 295

Describe the ER?

Answer 295

ER spreads throughout the cell, unlike in many diagrams The ER is a dynamic network that is continuously breaking and reforming The ER is connected to the nuclear envelope ER forms hollow tubes and flattened sacs. The chambers are cisternae Two types of ER: - Rough (RER) outer membrane covered in ribosomes - Smooth (SER) no ribosomes

Question 296

What associates with rough ER?

Answer 296

Ribosomes

Question 297

What are the functions of the endoplasmic reticulum?

Answer 297

Quality control Synthesis (e.g. proteins & lipids) Storage (e.g. steroid hormones) Detoxification (e.g. liver ER for alcohol)

Question 298

Describe the ERs function in quality control?

Answer 298

- Newly made proteins from ribosomes are fed through the translocon into the lumen of the ER and chaperone proteins associates with the protein to help it fold into its tertiary or quaternary structure - Disulfide bonds in transmembrane proteins are set up in the lumen of the ER as it is a non-reducing environment - If a protein is very mis-folded, it is sent out of the cell by reverse translocation into the cytosol and gets modified by ubiquitin which acts as a signal to break the protein down

Question 299

Describe the smooth ER?

Answer 299

-No ribosomes associated with the SER The SER is responsible for - ``` Phospholipid and cholesterol synthesis Steroid hormone production Synthesis and storage of glycerides Synthesis and storage of glycogen Important role as a calcium store ```

Question 300

Describe calcium signalling in Acinar Cells?

Answer 300

Zymogen granules contain enzymes important for digestion Stimulation -> Ca2+ release -> vesicle fusion -> enzyme release

Question 301

Describe transport from the ER to Golgi?

Answer 301

Transport between ER and Golgi in the form of vesicles and tubules The vesicles bud off the ER and are received by the Golgi Anything in the lumen or the membrane of the vesicle will be transported to the new compartment Both vesicles and tubules have a high SA : volume ratio which increases efficiency of transport The circles in which the cargo is being moved are the buds This process reduces leakage of material Asymmetry of bilayer is maintained when you have fusion

Question 302

How is cargo is incorporated into coated vesicles?

Answer 302

Coated vesicles form at exit sites and help with cargo selection, where they capture material to be moved to the right destination Different coats work differently

Question 303

What are vesicles coated with?

Answer 303

- Coat of specialised proteins - The coat aids formation of the vesicle, but must be discarded before the vesicle can fuse with the target compartment - Three types of vesicle coat (Coats must be removed from the membrane to prevent it becoming too bulky and this removal reveals a special family of proteins)

Question 304

How do vesicles reach the correct target ?

Answer 304

SNARE - soluble N-ethylmaleimide-sensitive factor adaptor protein receptor

Question 305

What are the two types of SNAREs?

Answer 305

v-SNAREs : vesicle SNAREs found in the vesicle membrane t-SNARE: target SNARES found in the membrane of the target membrane

Question 306

What do SNAREs do?

Answer 306

-When vesicles bud off they need to fuse with the right target and this is aided by SNAREs - SNAREs are integral membrane proteins - These are vital in ensuring the right vesicle fuses with the right membrane In nerve terminals, the SNARE complex involved in docking of synaptic vesicles is a helical bundle consisting of 3 components

Question 307

Describe the Golgi apparatus?

Answer 307

- Golgi apparatus is composed of flattened discs – also called cisternae (typically 5-6) - The Golgi apparatus tends to lie near the nucleus - The cisternae communicate with the ER and cell membrane by use of vesicles and tubules

Question 308

What are the 3 primary functions of the Golgi apparatus? (3)

Answer 308

1) Modification and packaging of secreted proteins 2) Renewal and modification of the plasma membrane 3) Delivery of material to other organelles, especially the endocytic pathway

Question 309

How does the structure of the Golgi appear relate to modifcation?

Answer 309

Many modification processes take place in the Golgi apparatus. Each modification process takes place in a specific region.

Question 310

What is the purpose of the Trans-Golgi Network?

Answer 310

Major sorting station for newly-made proteins

Question 311

How is plasma membrane and secreted proteins delivered to the cell surface by the constitutive secretory pathway in non-polarised cells through Trans-Golgi Network?

Answer 311

1) Signal mediated diversion to lysosomes = packaged into vesicles and recognised by a coat so lysosomal proteins end up in the right place 2) Signal Mediated diversion to vesicles = packaged into vesicles which are stored, proteins require signal, stored until it needs to be released by the cell 3) Constitutive secretion = movement of protein delivered to the cell surface, not regulated by signals

Question 312

What is taken up though endocytosis? (7)

Answer 312

``` Nutrients Signals Antibodies Enzymes Viruses Bacteria Membrane ```

Question 313

What different fates can endocytosed material have?

Answer 313

- Material taken up from endocytosis and immediately recycled to cell surface - Material can be delivered to lysosome that break materials down and allow It to be used by cell -Can be transcytosded, goes through lysosome and gets exocytosed though other side\ Need to avoid being degraded

Question 314

Name two endocytic pathways? (2)

Answer 314

Macropinocytosis Phagocytosis

Question 315

Describe the endocytic pathway macropinocytosis?

Answer 315

-Vesicle formed is much bigger, actin dependent process, important for uptake of nutrients in cancer cells (major way to gain nutrients), adapted as it is more selective, low SA to V ratio so can take up lots of extracellular fluid, important in immune cells, need to take up lots of fluid to gauge whether or not to produce immune response

Question 316

Describe the endocytic pathway phagocytosis?

Answer 316

The was bacteria and dead cells are engulfed (uptake of large particles)

Question 317

How are bacteria engulfed by phagocytosis?

Answer 317

- Bacteria often coated with lots of antibody molecules when they enter our cell - Antibody molecules bind to receptors - Macrophage extends pseudopod, initiates signals within cell and mobilizes actin cytoskeleton that forms arms to engulf bacteria, surrounded by pseudopods which fuse and form phahososme

Question 318

How are yeast cells engulfed by phagocytosis?

Answer 318

- Done by phagocytosis and micropinocytosis | - GFP tagged protein engulfed (yeast)

Question 319

Describe how macropinocytosis works?

Answer 319

- Cells form actin driven ruffles which sometimes fuse to form macropinosomes - Mechanistically similar to phagocytosis - Non-selective uptake of extracellular material - Used by cancer cells to take up nutrients

Question 320

Describe clathrin-mediated endocytosis?

Answer 320

- Cholesterol travels in low density lipoprotein (LDL) - Cells recognize LDL receptors can still bind in low amounts as very sensitive - When bind they cluster into microdomains known as coated pits - Becomes more curved and forms coated vesicle - Uncoating removed coat - Fuses with endosome - Separation of LDL with receptor put into vesicle which can re-enter through recycling pathway

Question 321

What is dynamin required for?

Answer 321

Dynamin is required to pinch off clathrin coated vesicles

Question 321

What is dynamin required for?

Answer 321

Dynamin is required to pinch off clathrin coated vesicles: Dynamin pinches of vesicles Clatherin invaginations are halted before they can pinch off in mutations Paralysis occurs because can’t recycle membrane

Question 322

Why is gradient of pH key for function on the endocytic pathway?

Answer 322

Viruses delivered through endocytic pathway need reduced PH to go through fusion with membrane Organelles refined by particular RAB (small GTPases) proteins, Important for defining cellular identity RAB 5 contributes to the specificity of fusion and ensure that fusion occurs efficiently

Question 323

What is cargo for degradation incorporated into?

Answer 323

intraluminal vesicles (ILVs).

Question 324

What is the Multivesicular Body (MVB)

Answer 324

``` Multivesicular body (MVB) - Limited membrane vesicles have budded from membrane inwards Work at certain PH again (Late endosome) ```

Question 325

What are lysosome important for?

Answer 325

Act as hydrolases and lipases Lysosomes also important in cell signaling (Lysosomes stained with acid phosphatase)

Question 326

What are mitochondrial and proximal proteins coded by?

Answer 326

nuclear genome

Question 327

What do the mitochondria do?

Answer 327

Energy production | Mitochondria make large amounts of ATP

Question 328

Describe mitochondria structure?

Answer 328

Mitochondria have a double membrane Outer membrane encloses the organelle The inner membrane is highly folded (high SA ). The folds are known as cristae The inner matrix contains the enzymes responsible for energy production

Question 328

Describe mitochondria structure?

Answer 328

Mitochondria have a double membrane Outer membrane encloses the organelle The inner membrane is highly folded (high SA ). The folds are known as cristae The inner matrix contains the enzymes responsible for energy production

Question 329

Describe mitochondria structure?

Answer 329

Mitochondria have a double membrane Outer membrane encloses the organelle The inner membrane is highly folded (high SA ). The folds are known as cristae The inner matrix contains the enzymes responsible for energy production

Question 330

What are adaptations of the mitochondrial outer membrane?

Answer 330

Perforated with large channels (Porins). These allow entry of molecules < 5000 kDa Contains enzymes involved in mitochondrial lipid synthesis

Question 331

What are adaptations of the mitochondrial intermembrane space?

Answer 331

Contains enzymes that use ATP to phosphorylate other nucleotides. H+ is pumped into this space (to create the proton gradient to drive Ox phos).

Question 332

What are adaptations of the mitochondrial inner membrane?

Answer 332

Folded into christae – this maximizes surface area. Contains the REDOX performing proteins of the electron transport chain. Proteins for ATP synthesis. Transport proteins to move molecules in and out of the matrix

Question 333

What are adaptations of the mitochondrial matrix?

Answer 333

Internal space containing enzymes of the Krebs cycle Contains: ``` Mitochondrial DNA Ribosomes tRNAs Enzymes (TCA, b-oxidation) Metabolites (e.g., TCA urea cycle, Ca++, K+, Mg++) ```

Question 334

what kind of genetic material do mitochondria contain?

Answer 334

The mitochondria contain their own genetic material Circular chromosome (double stranded) apprx 15-17kbps (bacterial plasmids are 744bp -2.58Mb, average = 80kb) Encodes 37 genes The mitochondrial DNA is inherited from the mother

Question 335

Describe the mitochondria life cycle?

Answer 335

Lots of damaging chemicals about so mitos have to have a mech to deal with this Mitochondria undergo fusion, fission etc to maintain integrity from damage ALSO they are reduced in number during mitosis – so have to recover When split up one half is destroyed the other continues as normal mitochondria, replicate by fission (mitochondria arises from existing mitochondria)

Question 336

How do you get proteins into ER?

Answer 336

Proteins translocate as they are synthesised ie unfolded

Question 337

What is the difference between getting proteins into mitochondria compared to ER?

Answer 337

The proteins are fully synthesised and then are translocated into the mitochondria. Still uses signal sequences Uses translocation proteins embedded in the outer and inner mito membrane

Question 338

What are the translocation proteins used to get proteins into mitochondria? (4)

Answer 338

TOM: Translocator of the Outer Membrane SAM: Sorting and Assembly Machinery TIM: Translocator of the Inner Membrane OXA: Cytochrome OXidase acitivity

Question 339

How do you get proteins into mitochondrial matrix?

Answer 339

N-terminal signal sequence – recognised by the TOM complex The protein translocates through TOM and TIM23 Translocates through TIM23 into matrix Signal is cleaved off

Question 340

How are mitochondrial Precursor Proteins Imported as Unfolded Polypeptide Chains?

Answer 340

The proteins could fold before docking with the TOM complex How do you stop this? - You bind interacting proteins to the newly synthesized polypeptide chain e.g., chaperones - ie proteins imported into mitochondria UNFOLDED.

Question 341

How are chaperones dissociated from the polypeptide chain?

Answer 341

Chaperones need energy (ATP) to dissociate the chaperones from the polypeptide chain The signal sequence is +ve charged The electrochemical H+ gradient driven by electron transport has two effects - ATP production - Membrane potential drives the +ve charged signal sequence through the IMM

Question 342

How do you get proteins into mitochondrial outer membrane?

Answer 342

Major proteins in OMM (outer mitochondrial membrane) are called porins They are beta-barrel proteins Problem: TOM cannot insert proteins into bilayers Solution: Enter in intermembrane space, kept unfolded by Chaperones SAM complex inserts and folds

Question 343

How do you get proteins into mitochondrial inner membrane?

Answer 343

Most common route For the Inner Mitochondrial Membrane: Uses TOM and TIM23 2nd route Protein completely enters matrix space Signal sequence cleavage unmasks a 2nd signal that causes insertion into OXA complex

Question 344

How is getting proteins into mitochondrial inner membrane and intermembrane space is varied?

Answer 344

Multi-pass IMM proteins Snake through TOM as a loop This allows chaperones to bind to stop folding and guide to wards TIM22

Question 345

How do you get soluble protein in the intermembrane mitochondrial space?

Answer 345

Cleave after membrane insertion

Question 346

Describe the structure of peroxisomes?

Answer 346

Peroxisomes only have a single membrane Do not contain DNA or ribosomes Found in all eukaryotic cells and carry out oxidative reactions Thought to be the remnant of an organelle found in the ancestors of eukaryotic cells

Question 347

What is the function of peroxisomes?

Answer 347

Performs variety of oxidative reactions Contain a variety of oxidative enzymes – catalase and urate oxidase Function to remove hydrogen atoms from various organic compounds according to the reaction

Question 347

What is the function of peroxisomes?

Answer 347

Performs variety of oxidative reactions Contain a variety of oxidative enzymes – catalase and urate oxidase Function to remove hydrogen atoms from various organic compounds according to the reaction

Question 348

Where are proximal membrane proteins made?

Answer 348

Most peroxisomal membrane proteins are made in the cytosol and then insert into the membrane of pre-existing peroxisomes.

Question 349

How do new peroxisomes arise?

Answer 349

New peroxisomes arise from preexisting ones, by organelle growth and fission ie like mitochondria

Question 349

How do new peroxisomes arise?

Answer 349

New peroxisomes arise from preexisting ones, by organelle growth and fission ie like mitochondria

Question 350

What is cell motility?

Answer 350

Cell movement

Question 351

What does cell motility need? (2)

Answer 351

Energy | Guidance

Question 352

What are microtubules?

Answer 352

Hollow tubes of a and b tubulin Circular shape provides support and rigidity, hollow structure

Question 353

What Microtubule-Based structures provide motility?

Answer 353

Cilia and flagella

Question 354

Describe Cilia and Flagella Structure

Answer 354

Same structure in cilia and flagella Flagella is a lot longer than cilia, similar cross section Major functional structure = The Axoneme

Question 355

Describe the structure of the Axoneme?

Answer 355

9 microtubule outer doublets One inner pair (2) Complete fibers have 13 protofilaments Outer and Inner arms carry out different functions Dynein arms are structure proteins that allow microtubule to move

Question 356

What is dynein?

Answer 356

Domain associated with microtubules, and a motor protein domain Doublets slide past each other when ATP produced

Question 357

How do we convert sliding motion in dynein into bending motion?

Answer 357

Nexin Crosslinkers When we apply energy sliding generated by dynein, we add flection to bend the flagella

Question 358

What is the difference between cilia and flagella movement?

Answer 358

Cilia stays straight up and waves while flagella is more of a whip crack wave

Question 359

Describe the components of cilia and flagella movement?

Answer 359

- Outer arm generates power - Inner arm generates waveform - Dynein controls arm movement

Question 360

What are centrosomes made up of?

Answer 360

pair of centrioles

Question 361

Describe actin filament structure?

Answer 361

Each actin filament has a plus and a minus end, and motor proteins like myosin 'walk' directionally along the filaments. Actin filaments can be arranged in bundles or networks using various kinds of crosslinking proteins. The bundles and networks are important for muscle contraction, cell shape, and cell adhesion.

Question 362

Describe assembly of a polarised filament? (Actin)

Answer 362

Plus fast growing end and stable minus end Barbed-end(+) Pointed-end(-)

Question 363

Describe actin-binding protein profilin?

Answer 363

Profilin (inhibits nucleation) stops actin (binds to atp actin and accessory proteins regulate actin dynamics) from binding to each other, so we don’t see polymerization (severe action of Cofilin)

Question 364

What are the types of actin-binding proteins?

Answer 364

1) Monomer nucleating 2) Monomer sequestering 3) End-blocking (capping) 4) Monomer polymerising 5) Depolymerising 6) Bundling 7) Filament-severing 8) Membrane-binding

Question 365

What is myosin?

Answer 365

The Microfilament Motor Protein

Question 366

What is the filament and motor of Cilia/flagella and Cytoskeleton/muscle?

Answer 366

_________ - Filament - motor Cilia/flagella - Microtubule - Dynein Cytoskeleton/muscle - Actin - Myosin

Question 367

Describe actin-based motility with Filopodia, Lamellipodia, Stress fibres and cortical actin? (movement of the cell using different parts)

Answer 367

Filopodium-narrow finger like projection from the cell, not a strong structure that drives movement, used more to explore surrounding and provide a pioneering point. Lamellipodium- mesh work of actin filaments, pushing forward the front of the cell forming a lamellipodium, soft and wide. Stress fibres- although lamella push forwards the front, we need to translocate the body as well for cell migration, actin is providing the force that is going to drag the whole cell body behind the moving front. Cortical actin sheath- an envelope around the cell, beware of other actin structure we can come across.

Question 368

How do we see migration in actin-based motility?

Answer 368

If we want to see migration, we want it to be in response to a stimulus, we might provide a growth factor or a cytokine. This is going to provide direction & guidance which will form the filopodium.

Question 369

What does the ECM (extra cellular matrix) regulate? (4)

Answer 369

- Migration - Tissue integrity and cell shape - Proliferation - Differntitation

Question 370

Describe the appearance of the ECM

Answer 370

3D meshwork of extracellular matrix. Cells integrated into the matrix. Presence of elastic fiber allows skin to stretch. Hyaluronan, proteoglycans and glycoproteins fill in the space

Question 371

What are the types of ECM? (3)

Answer 371

Fibrous Proteins (structural) - Collagens - Elastin Adhesion Proteins (interact with cells) - Fibronectin - Laminin Hydrated Macromolecules - Glycosaminoglycans (Gags) - Proteoglycans (Protein + Gags)

Question 372

What is the main ECM component?

Answer 372

Collagen

Question 373

Describe the structure of collagen?

Answer 373

Triple Helix Glycine-proline-hydroxyproline triplet repeats 3 a Chains

Question 374

What are collagens produced by?

Answer 374

fibroblasts and epithelial cells

Question 375

Describe collagen synthesis?

Answer 375

Secreted by fibre glass and epithelial cells. At the top there is modifications. Lack of vitamin C will lead to defects of collagen, as vitamin C is a cofactor of hydroxylation. Most of the assembly is self-assembly. Self assembly of collagen fibril.

Question 376

Describe the features of elastin?

Answer 376

Retains its integrity as it extends. As we stretch the elastin it gains structure, it maintains it integrity. Elastin made of tropoelastin, tropoelastin is oxidised by lysyl oxidase to make elastin. Makes up 50% of the dry weight of our aorta

Question 377

What is Fibrillin?

Answer 377

Structural protein. The scaffold over which elastin is laid down.

Question 378

Describe GAG's Glycosaminoglycans?

Answer 378

Largely made up of disaccharide chains. Highly charged partly because of acidic groups, and hydroxyl groups, and highly sulfated. Makes the molecules very hydrophobic. A way of holding large amount of water in a tissue. In a wound healing response, want to have cells migrating, need room to move through something, if we introduce sugars that will draw water in and it will expand creating space for our immune cells to migrate. Where they differ is in the GAG chain. 80 saccharides in the disaccharide chain. The sugar will typically be conjugated to a serine residue, through a conserved linking tetrasaccharide. Sugars take up a lot of space.

Question 379

Describe Hyaluronan Complexes?

Answer 379

Not sulfated but still highly charged. It’s big. Around 25,000 sugars long. Keratan sulfate, and chondroitin sulfate is a sugar chain, substituted onto a protein (aggrecan) are linked to the hyaluronan core through a linker molecule. Hyaluronan is good at retaining water. Forming the eye, if we introduce hyaluronan that we want to be hollow, it draws in a lot of water. We will see an empty space, then see cell layers migrating around the inner side of that hollow dome, forming the structure of the eye. Once it is formed the hyaluronan is digested and you are left with a hollow structure. Proteoglycans capture soluble ligands.

Question 380

Describe what performs the function of Cushioning? Space filling? Signal binding?

Answer 380

Cushioning- fulfilled by sugars by recruiting water Space filling- fulfilled by sugars Signal binding- sugars can act as a way of signal binding as well as ECM

Question 381

Describe Adhesion Glycoproteins: Laminin?

Answer 381

Epithelium at the top of the skin. A flat layer. Polarised. Nutrients absorbed by the gut move in one direction due to polarisation. Polarity achieved by the basal lamina. Basil lamina is our adhesive extracellular matrix protein. A tough mat of protein that our epithelium sits on. Laminin wants to be interacting with things. Self assembly domains. Sites that will interact with other ECM proteins, Nidogen. Binding sites via integrin. If cells are going to stick to extracellular matrix it will be through integrins. Our adhesive extracellular matrix proteins will have integrin binding sites.

Question 382

Describe Adhesion Glycoproteins: Fibronectin?

Answer 382

-Acts as adhesive signalling protein for collagen network Cross linking sites, disulphide bonds, to form a network. Self association sites, spontaneously organise itself. Binding cells for other ECM proteins, collagen binding, will have a collagen network that would then be coated with fibronectin as our adhesive signalling protein. Cell binding domain, how the fibronectin will interact with integrins and therefore facilitate cell adhesion. Very repetitive globular protein. Type-3 repeats, in repeats 9 and 10 we see the RGD sequence, which is our integrin binding motif, (Arg positively charged, Gly negatively charged, Asp negatively charged). Charged group, interacts well with charged molecules.

Question 383

What do interns do?

Answer 383

Bind matrix through divalent cations- introducing a large amount of charge, will bind to charge residues Removal of cations causes cells to detach without damaging anything

Question 384

What are Focal Adhesions

Answer 384

Adhesions that we find at the termini of actin stress fibres that are going to bind to the ECM. Integrin be transmembrane receptors that form the link. These are going to link to the actin cytoskeleton.

Question 385

What are the components of focal adhesion? (3)

Answer 385

Alpha, beta- integrin dimer, transmembrane receptor. Forging the link of the integrins. Whole battery of cytoskeletal proteins that play a structural role that binds integrin to actin-myosin contractile operators. Talin- bind to cytoplasmic domain of the integrin, contractions actin binding sites, and introduces other proteins. Adhesion dependent growth- signalling proteins, focal adhesion kinase, activated upon adhesion. (-Transmembrane receptor) (-Connection to cytoskeleton) (-Signalling)

Question 386

What are the knockouts of focal adhesion proteins?

Answer 386

``` β1-integrin Embyonic lethal at implantation (Day 5) α5-integrin α5β1-integrin fibronectin receptor Embyonic lethal mesoderm development (Day 10) Fibronectin Embryonic lethal (Day 9) Talin Embryonic lethal (Day 6-8) ```

Question 387

What are some integrin-related defects?

Answer 387

αIIbβ3-integrin - Platelets bind fibrinogen to clot blood - Glanzmann’s thrombasthenia - Bleeding gums and nose bleeds β2-integrin Leukocyte Adhesion Deficiency (LAD) syndrome Impaired expression Recurrent bacterial infections

Question 387

What are some integrin-related defects?

Answer 387

αIIbβ3-integrin - Platelets bind fibrinogen to clot blood - Glanzmann’s thrombasthenia - Bleeding gums and nose bleeds β2-integrin Leukocyte Adhesion Deficiency (LAD) syndrome Impaired expression Recurrent bacterial infections

Question 388

What are the layers in a cell?

Answer 388

Apical (epithelial) | Basal (basal lamina)

Question 389

Describe the anchors in the basal lamina layers of a cell?

Answer 389

Actin-linked cell-matrix adhesion anchors bind actin filaments in cell to extracellular matrix hemidesmosome anchors intermediate filament in a cell to extracellular matrix

Question 390

What are adherens junctions also known as?

Answer 390

Belt desmosomes

Question 391

Where are actin filaments found?

Answer 391

inside microvillus

Question 392

What are the two types of actin-linked junctions? (2)

Answer 392

Adherens junctions | Focal adhesions

Question 393

What do Focal Adhesions and Adherens Junctions link to?

Answer 393

The actin cytoskeleton

Question 394

What do hemidesmosomes (half a desmosome) do?

Answer 394

- Attach Cells To Basal Lamina | - Integrins And Intermediate Filaments

Question 395

What do desmosomes do?

Answer 395

Cell - Cell Junctions Cadherins And Intermediate Filaments Plentiful in heart muscle and epidermis

Question 396

What is the function of focal adhesions?

Answer 396

Way to attach cells to basal lamina

Question 397

# Fill in the table? ________ - actin-linked junctions- Intermediate filament Intergrin - Cadherin -

Answer 397

________ - actin - Intermediate filament Intergrin - Focal adhesion - Hemisdesomosome Cadherin - Adherens junction - Desmosome

Question 398

What does desmoglein do?

Answer 398

Hold together keratinocytes in epidermis ( that's why pemphigus causes autoimmune skin bleeding as causes desmoglein to be targeted)

Question 399

What do tight junctions do?

Answer 399

- To Prevent Fluid, Ion and Membrane Flow - Variable Extent Allow: - Transcellular Transport (substances travel through the cell) - Paracellular Transport (substances travel intercellular space between the cells)

Question 400

What are the major tight junction proteins? (2)

Answer 400

- Occuludin and claudin | - 1 type of occludin and 25 types of claudin

Question 401

What binds to claudin, occludin and actin?

Answer 401

Zonula occludens proteins

Question 402

How are lipids and proteins segregated?

Answer 402

Apical Outer Membrane - Glycolipid - Cholesterol Basolateral -Phosphatidylcholine

Question 402

How are lipids and proteins segregated?

Answer 402

Apical Outer Membrane - Glycolipid - Cholesterol Basolateral -Phosphatidylcholine

Question 403

What do gap junctions allow?

Answer 403

Allows regulated and direct cell-cell communication

Question 404

What is contained in gap junctions?

Answer 404

Water, inorganic ions, sugars, amino acids, ATP, cAMP, IP3

Question 405

Describe gap junctions?

Answer 405

-Fairly big clusters -Need membranes to be very close together -Close apposition of membranes: gap = 2- 4 nm Wide distribution -Connective tissue, epithelia, neurons, heart muscle -Form 1.5 nm diameter pores

Question 406

Describe the gap junction structure?

Answer 406

Gap junctions made of connexins 6 connexins forming single connexon Different arrangement and types Potential to generate a lot of different gap junction types

Question 407

Describe Gap Junction Regulation

Answer 407

- When we damage neuron membrane depolarizes, PH changes acts as protection mechanism - Responds to damage, prevents mass death of cells - Important to limit damage caused by calcium influx - If we damage neuron, other connected neurons will die (-Calcium important in cellular functions but is also toxic)

Question 408

Describe the order of structures in a cell-cell junction?

Answer 408

- Tight junction - Adherens junction - Desmosome - Gap junction - Hemidesmosome

Question 409

What are the 3 critical events of the cell cycle? (3)

Answer 409

1) chromosome replication 2) chromosome segregation 3) cell division

Question 410

Explain the clock movement of the cell cycle?

Answer 410

- Existence of a master governor that makes major decision regarding cell fate = cell cycle clock, which operates in the nucleus - Can slow down stop and go backwards - Cells need stimuli, external signals to tell cell signal to proceed (signalling proteins can overrule stimuli)

Question 411

What is cell proliferation?

Answer 411

cell replication

Question 412

Describe the sub-phases of mitosis in cell division?

Answer 412

Interphase Prophase: Movement of the centrioles to polar end of the nucleus; condensation of chromosomes Prometaphase: Components of the mitotic spindle elongate away from the spindle poles, allows chromosomes to align and separate properly into daughter cells Metaphase: Chromosome alignment is complete Anaphase: Pairs of sister chromatids are separated Telophase: Chromosome start to de-condense, nuclear membrane starts to be re-established. Cytokinesis

Question 413

How do you remember the mitosis sub-phases?

Answer 413

``` In - Interphase Peaceful - Prophase Pakistan - Pro-metaphase Many - Metaphase Are - Anaphase Terrorists - Telophase Caboom - Cytokinesis ```

Question 414

Describe the stages of the cell cycle?

Answer 414

INTERPHASE: main part of cell cycle 1) G1 phase - cell increases size - ribosomes, RNA produces - preparation for DNA synthesis 2) S phase -DNA synthesised (chromosomes duplicated) 3) G2 phase - cell checks fidelity of DNA - preparation for nuclear division Mitosis: cell division (prophase, pro-metaphase , metaphase, anaphase, telophase. cytokinesis)

Question 415

What happens during G1 phase?

Answer 415

Phase is where cells grow and perform their physiological function. Transit through G1 is driven by external signals (growth factors etc). Cells can exit G1 into G0, where they will not-divide and not grow. They can re-enter the cycle from G0 Restriction point, point of no return cell makes decision on whether or not it is under good condition to undergo replication, checks environmental conditions

Question 416

What is the restriction point in G1 phase?

Answer 416

Restriction point, point of no return cell makes decision on whether or not it is under good condition to undergo replication, checks environmental conditions

Question 417

What are checkpoints in the cell cycle?

Answer 417

Breaks in the cycle to make sure everything is going well Places in the cell cycle where cell is monitoring things Allows cells to increase length of particular phase to correct errors Checks form damaged DNA , monitors whether replication forks are stalling due to problem or not (facilitates repair)

Question 418

What is the G1: growth v/s quiescence decision?

Answer 418

Discrete window to consult the extracellular environment: from the onset of G1 phase to an hour or 2 before the G1-to-S transition. G1 decision making machinery apparent in the responses of cultured cells to extracellular signals: Serum and growth factors removed before the cells have completed 80-90% of G1 -> fail to proceed further and revert to G0 state Serum and growth factors removed in the final hr of G1 -> proceed to S, G2 and M phase

Question 418

What is the G1: growth v/s quiescence decision?

Answer 418

Discrete window to consult the extracellular environment: from the onset of G1 phase to an hour or 2 before the G1-to-S transition. G1 decision making machinery apparent in the responses of cultured cells to extracellular signals: Serum and growth factors removed before the cells have completed 80-90% of G1 -> fail to proceed further and revert to G0 state Serum and growth factors removed in the final hr of G1 -> proceed to S, G2 and M phase

Question 419

How does cancer relate to restriction point?

Answer 419

Restriction point is what goes wrong in a lot of cancer cells, cell is blind to restriction point so carry-on replicating

Question 420

How do we find a gene or genes responsible for bringing about a cell cycle transition?

Answer 420

Genetic approach: Requires cells that have a mutation in a putative cell cycle transition gene Biochemical approach: Requires supply of large numbers of cells undertaking the same transition at the same time (How long-lived is the signal?)

Question 421

Why is Yeast as a genetic model for cell cycle?

Answer 421

-You can tell which phase of the cell cycle the yeast cell is in by just looking Can look at size and morphology to cell which phase it is in Can grow yeast as haploid or diploid organism If turned into haploid, some with mutation you can get large numbers to see phenotype of mutation

Question 422

How can you test mitosis without a cell?

Answer 422

- Can put cytoplasm in test tube - One can deplete the cytoplasm of different proteins using antibodies -One can remove cytoplasm at different stages to study changes (eg in protein phosphorylation) over time.

Question 423

How do we isolate the proteins that are responsible for cell cycle transitions?

Answer 423

Frogs' eggs grow arrested in G2 to a relatively large size (good for extracting decent amounts of protein) Oocytes arrested in G2, then under hormonal control mature and start meiosis BUT when they are laid, they become stuck in meiotic metaphase i.e., M-phase. The are released by fertilisation -> to start dividing

Question 424

Something in the frog Egg cytoplasm can catalyse the transition from G2 to M-phase, what was it?

Answer 424

The factor was called Maturation Promoting Factor (MPF)

Question 425

What is MPF (maturation promoting factor) produced by?

Answer 425

MPF produced by support cells, spike near meiosis, metaphase arrest and during cell division events cell cleavage All spike during these areas This factor is driving the cell cylce

Question 426

What triggers the G2 to M transition?

Answer 426

This identified Cyclin-dependent kinases (CDKs) and Cyclins Protein sequence of these two proteins analyzed Sequence was found to be similar to protein kinases

Question 427

What do protein kinases do?

Answer 427

Protein kinases are signaling devices which operate to create molecular switches Kinases adds target protein to phosphate (uses ATP to get phosphate) phosphorates can activate certain target proteins Different kinases turned on and off in different phases of the cycle to activate certain proteins

Question 428

How do Cell cycle transitions involve the irreversible destruction of cyclins?

Answer 428

- Kinases were there all the time but not active all the time, inactive kinase bind to cyclin protein which activates the kinase - Kinase turned off by destruction of cyclin

Question 429

What is the one cell cycle regulator gene in yeast called?

Answer 429

Cdk1

Question 430

What are the cell cycle regulator genes in mammals?

Answer 430

- Different Cdk1s and different cyclins in mammals - Cyclins grouped based on different phases of the cell cycle - Cyclins destroyed as enters s phase, then m-cdks have expression increased and bind to their cyclin etc…

Question 431

How do levels of different cyclins differ in the cell cycle?

Answer 431

Cyclin E: low levels throughout most of G1, rapid increase after the R point Cyclin A: levels increase in concert with the entrance in S phase Cyclin B: levels increase in anticipation of mitosis Collapse of cyclin levels as the cell progresses through the cell cycle -> degradation (ubiquitination-dependent) This means the cell cycle can only progress in 1 direction

Question 432

What are D-type cyclins are controlled by?

Answer 432

extracellular signals

Question 433

What are Cyclin/CDKs are regulated by?

Answer 433

CDK inhibitors (CKIs)

Question 434

What is a chromosome?

Answer 434

linear DNA molecule

Question 435

What is a centromere?

Answer 435

Region where the spindle attaches

Question 436

What are homologous chromosomes?

Answer 436

Chromosomes that have the ‘same’ genes arranged in the same order 1 Inherited from father, 1 from mother

Question 437

What are chromatids?

Answer 437

Newly copied DNA strands still joined to each other by a centromere

Question 438

What does M-Cdk trigger?

Answer 438

Majors changes to get ready before M-phase - Assembly of the mitotic spindle - Each sister chromatid is attached to an opposite pole - Chromosome condensation - Breakdown of the nuclear envelope - Rearrangement of the actin cytoskeleton + Golgi

Question 439

How does M-cyclin/Cdk triggers entry into mitosis?

Answer 439

- Cyclin increased in expression and binds to M-Cdk (it’s still inactive). - CAK (Cdk-activating kinase) adds a phosphate group and activates M-Cdk. - Wee1 adds an inhibitory phosphate that inactives M-Cdk. - Cdc25 (phosphatase) gets activated and acts on the M-Cdk, it removes the inhibitory phosphate, and activates the M-Cdk. - Positive feed back, active M-Cdk and drive activation reaction of Cdc25. Also, positive feed back of Cdk-inhibitory kinase when levels of active M-Cdk are high. - Process of activation begins with S-Cdk complexes, start to act on Cdc25. - In late G2 the Cdc25 phosphatase is triggered to activate a positive feedback loop rapidly activating mitosis.

Question 440

Explain the APC (Anaphase-Promoting Complex)?

Answer 440

Progression trough metaphase/anaphase transition -> driven by protein destruction 2 targets: 1) S+M cyclins: if these are destroyed most CDKs are inactivated -> CDK targets are Dephosphorylated (by phosphatases) APC/C kept on in early G1 -> turned off as G1/S-CDK activated to allow Cyclin accumulation (2) Securin: protects the protein linkages that hold sister chromatids together Destruction -> activates a protease that separates the sister chromatids -> anaphase

Question 441

How does APC (anaphase-promoting complex) contrast to phosphorylation?

Answer 441

Contrasts to phosphorylation which used activate/inhibit proteins such as M-cdk

Question 442

What can go go wrong in mitosis?

Answer 442

Most genes that you have, need mutations in both alleles to cause a phenotypic change. If you were just heterozygous, if you had one mutant allele and one normal the cell can still function. Familial- if you inherit one faulty copy of a gene, and you get a mutation in the second allele, the phenotype will then start to be expressed, loss-of-heterozygosity. Sporadic- no inheritance of faulty copy, you have two somatic mutations. You can lose a chromosome so you only have one copy, hemizygosity, and if it is mutant then the mutant phenotype will show. Lagging chromosome- chromosome ends up in the wrong place, it stays in the wrong daughter cell. This is called chromosome non-disjunction.

Question 443

Describe the Basic Structure of the Mitotic spindle

Answer 443

Astral microtubules - glued to the outside of the cell Kinetochore microtubules - microtubules that attach to the centromeres of the chromosomes Interpolar microtubules - overlap, part of the process by which the cell and chromosome is going to be achieved

Question 444

How can you tell successful mitosis has taken place?

Answer 444

sister chromatids going to opposite poles | Trail and error is used to get 1 kinetochore to spindle pole

Question 445

How do cells make sure spindle is stable?

Answer 445

- They contact their kinetochores. - By trial and error, you get the correct stable configuration, one kinetochore attached to each pole. - Cells have mechanism which sense the tension by when the kinetochores attach to the spindle to make sure it’s stable. - What happens in the incorrect attachments, the tension is lower and sensed, generates an inhibitory signal, which loosens the kinetochores attachment. LC

Question 446

Describe the activation of APC/C by Cdc20 leads to the ubiquitylation and destruction of securin?

Answer 446

APC/C activation, destroys the securin, and activates separase, chromatids then free to physically separate. Cdks can phosphorylate separase -> inhibition BUT in anaphase -> cyclins destroyed -> Cdk inactivation -> less separase phosphorylation -> activation

Question 447

What is the difference between anaphase A and anaphase B?

Answer 447

ANAPHASE A: kinetochore microtubules separate by shortening In ANAPHASE B the ASTRAL microtubules pull the cells apart by motors and depolymerisation. The interpolar microtubules slide past each other. It is in TELOPHASE that the nucleus (nuclear membrane etc) is reassembled and cytokinesis can begin KEY POINT: This is complex and can go wrong

Question 448

What does cell do if it senses it has the incorrect number of chromosomes?

Answer 448

Carries out apoptosis (cell death)

Question 449

Describe how cells die with loss-of-heterozygosity (LOH) by nondisjunction?

Answer 449

Loss of extra chromosome in attempt to repair it. Depending on which one you lose, depend if it has a good or bad consequence. One mutant chromosome and you’re fine. If you have two mutant alleles you have lost of heterozygosity, rendered a cell carrying a mutation.

Question 450

How does cell react to generation of hemizygosity (has one member of chromosome pair rather than two)?

Answer 450

Generation of hemizygosity, you get a cell hemizygous cell, and the cell is somehow viable and escapes apoptosis. Loss of one copy of the pairs of chromosomes. There is a difference in consequence of non-disjunction, when it happens in your adult mitotic cells and an embryo when you are developing. (catastrophic consequences in early embryogenesis).

Question 451

How does loss of heterozygosity (LOH) come about from mitotic recombination?

Answer 451

Mitotic recombination, can occasionally happen in mitosis. By swapping material about, you can end up with what’s on the right. Swapping, because of this recombination, you can generate a cell that is homozygous for the mutant allele (rare). Loss of heterozygosity

Question 452

How does loss of heterozygosity come about from gene conversion?

Answer 452

Polymerases start copying DNA stick to their strand, however sometimes you can get DNA very close to each other, the chromatids being formed are very close to each other which can cause the polymerases jumping from one DNA to another. You can accidently copy part of a green strand into a red strand.

Question 453

What are the ways mitosis can go wrong by inducing loss of heterozygosity (LOH)? (3)

Answer 453

- Non-disjunction - Mitotic recombination - Gene conversion

Question 454

Describe the process of meiosis?

Answer 454

Diploid organisms have two versions of each chromosome (homologues). Homologues are either paternal or maternal. Only one homologue for each chromosome is packaged into a gamete. Meiosis resembles mitosis except that there are extra steps that segregate homologous chromosomes. Pairing of homologues before segregation allows for crossing-over (homologous recombination). Two stages: Meiosis I and Meiosis II

Question 455

Explain crossing-over and segregation in meiosis 1?

Answer 455

Centrioles + chromosomes are replicated (just like mitosis) Maternal + paternal homologs pair up Genetic diversity is generated by recombination between homologous chromosomes One complete chromosome (2 chromatids) pulled to sep. poles Crossing-over takes place when homologues pair up.

Question 456

Describe the process of meiosis 2?

Answer 456

- Resembles mitosis | - The main difference is that cells in meiosis 2 are haploid rather than diploid

Question 457

Describe meiotic prophase 1?

Answer 457

- Lined up in bivalents - Process of homologous combination takes place, forms chiasma - Not only genetic recombination from pairing but also aligns chromosomes for anaphase - Pairing in facilitated by the synaptonemal complex (proteins) as well as DNA base pairing between homologues.

Question 458

What are the two purposes of meiotic prophase 1?

Answer 458

1) It aligns the chromosomes up ready for anaphase (along with the formation of the synaptonemal complex) 2) It allows for genetic recombination between paternal and maternal DNA on the same chromosome

Question 459

What leads to formation of synaptomental complex?

Answer 459

Chromosome Homologs pairing up

Question 460

What happens when chromosome homologs pair up?

Answer 460

Possibly a recombination complex (which recognises ds breaks) helps bind the homologs together The axial core (proteins that bind the chromatin via cohesion) are cross-linked by transverse filaments to form the Synaptonemal complex

Question 461

What does the tight bringing together of sister chromatids by the synaptonemal complex do?

Answer 461

- aligns the two chromosomes | - helps in homologous recombination

Question 462

What is the main difference between mitosis and meiosis?

Answer 462

Mitosis: Sister chromatids separate Meiosis: Homologs separate

Question 463

What allows homologs to separate in meiosis?

Answer 463

(i) BOTH kinetochores (on one chromosome) attach to the same spindle pole (in mitosis you to avoid this) This is done by a protein complex that is removed after meiosis I (ii) Crossing over = physical linkage between homologs (iii) Cohesin is only removed from the arms

Question 464

What does regulation in meiosis do?

Answer 464

- Ensures at least 1 crossover forms - Crossover interference – once one forms it inhibits others close by Thus the number per arm are limited

Question 465

How does meiosis go wrong, what are the two types of chromosome abnormalities?

Answer 465

- abnormalities in chromosome number | - chromosome structural rearrangements.

Question 466

What is Aneuploidy?

Answer 466

Unbalanced number of chromosomes | extra or 1 less

Question 467

How does Aneuploidy (incorrect chromosome number) happen?

Answer 467

Disorders of chromosome number are caused by chromosome non-disjunction Either - homologous chromosomes - sister chromatids fail to separate in Meiosis I Meiosis II Mitosis

Question 468

What are the consequences of numerical chromosomal abnormalities?

Answer 468

Usually lethal in some way, in sex chromosome on the rare chance of survival they are infertile?

Question 469

Why do we see lethality from syndromes arising from nondisjunction?

Answer 469

Two explanations Haploinsuficiency – pseudoautosomal genes are expressed from both alleles and dose matters Imprinted genes on X – ie monoallelic expression which is lost. ie null

Question 470

What are the two ways cells die? (2)

Answer 470

Necrosis (1 cell dies often induces surrounding cells to die) - reviews external message Apoptosis (very controlled form of cell death) - can be external or internal signal

Question 471

How does necrosis occur?

Answer 471

- Physical damage - Toxins (external e.g snake venom or internal e.g bacterial) - Hypoxia – is a low Oxygen concentration - Ischemia – Loss of blood supply – therefore you loose oxygen and nutrient supply

Question 472

When does apoptosis occur?

Answer 472

Physiological situations: - Tissue size maintenance - Developmental cell loss – growth factors - Removal of immune cells - Hormone-dependent involution - Inappropriate interactions - Anoikis Pathological situations: - DNA damage e.g. radiation, oxidative stress - Virally infected cells

Question 473

What are the characteristics of necrosis?

Answer 473

Reversible: - Membrane integrity compromised - Organelle and cell swelling Irreversible: - Increased intracellular calcium - Autolysis - Cell bursting (cell lysis) - Elicits an inflammatory response

Question 474

What is autolysis?

Answer 474

destruction of cells by their own enzymes

Question 475

What are the characteristics of apoptosis?

Answer 475

- Shrinkage - Nuclear breakdown - Apoptotic bodies - Phagocytosis - No inflammatory response - Requires energy - Controlled cell death - Relationship with autophagy

Question 476

What is apoptosis' relationship with autophagy?

Answer 476

Apoptotic bodies - vesicles containing dying parts of cells | Autophagy to try to rescue damaged cells

Question 477

Can necrosis and apoptosis occur at the same time?

Answer 477

In reality, both necrosis and apoptosis can occur in the same tissue Equally, they can occur separately An example would be brain ischaemia (cells in middle die of necrosis on edge die of apoptosis)

Question 478

How is apoptosis used in development?

Answer 478

Controlled cell death maintenance of tissue size and to prevent pathological conditions e.g Neuronal connections are refined by the competition for survival factors Not enough survival factor = apoptosis

Question 479

What do Ced genes do?

Answer 479

Ced genes involved from recognition of apoptotic signal to engulfment of apoptotic cell by phagocytes C-elegans provide an excellent model for studying apoptotic pathways

Question 480

What are caspases?

Answer 480

The executioners of cell death = essential for apoptosis ``` C = cysteine at their active site asp = aspartic acids are the cleavage site in target proteins ``` Irreversible pathway

Question 481

Describe initiator caspases?

Answer 481

- activated by apoptotic signals | - activate executioner caspases

Question 482

What do executioner caspases do?

Answer 482

Cleave over 1000 proteins

Question 483

How do you amplify proteolytic cascade?

Answer 483

one initiator caspase can activate multiple executioner caspases

Question 484

What are targets of caspase?

Answer 484

Cause breakdown of nucleus structure, including the nuclear lamina through cleavage of nuclear lamins Prevents DNA repair by cleaving the DNA repair enzyme PARP (Poly ADP-ribose polymerase) Cause cytoskeletal changes, for example the breakdown of actin, by cleaving cytoskeletal proteins like Gelsolin

Question 485

What are the two ways to activate apoptosis? (2)

Answer 485

1) Extrinsic pathway | 2) Intrinsic pathway

Question 486

Describe the extrinsic pathway to activate apoptosis?

Answer 486

- Tumor necrosis factor family - 6 related receptors – the death receptors - Indirectly (via the DISC) activate initiator caspases DISC: Death-inducing signalling complex This is a multi-protein complex formed by members of the "death receptor" family of apoptosis-inducing cellular receptors

Question 487

Describe the Intrinsic pathway to activate apoptosis?

Answer 487

Triggered by: - stress signals e.g. DNA damage - developmental signals When stimulated, release of cytochrome c into cytosome Leads to assembly of apoptosome from proteins such as Apaf1 and CARD This recruits caspase which can then be activated and used to cleave

Question 488

What decides whether cytochrome c or other intermembrane mitochondrial proteins are released in the intrinsic pathway of apoptosis activation?

Answer 488

Balance between pro- and anti-apoptotic factors known as Bcl2 family proteins - EGL-1 homologue BH3-only protein = pro-apoptotic - Ced9 homologue Bcl2 protein = anti-apoptotic

Question 489

What is cancer?

Answer 489

a disease of aberrant cell proliferation & differentiation

Question 490

What factors could be an environmental influencer in cancer?

Answer 490

Infection Diet Noxious agents

Question 491

How can diet act as an environmental influence on cancer?

Answer 491

Koji mold from food such as rice and peanuts can cause hepatocellular carcinoma This mold generates an aflatoxin, passes through and is modified by your liver Cytochrome p-459 enzymes modify molecules to make them more soluble In aflatoxin it is activated to make aflatoxin 2 3 epoxide This targets guanine in the causes DNA damage and mutations

Question 492

How can noxious substances act as an environmental influence on cancer?

Answer 492

Asbestos dust causes type of cancer Some conditions are caused by significant exposure to asbestos dust

Question 493

What causes inherited forms of cancer to arise?

Answer 493

Cancer is a consequence of chromosomal changes (e.g translocation) -Fusion of two genes may cause gene to be unable to switch off leading to constant proliferation

Question 494

What is an oncogene?

Answer 494

A gene with the potential to cause cancer by transforming | cellular behaviour

Question 495

How do oncogenes come about?

Answer 495

Arise from genes involved in regulated proliferation – PROTO-oncogenes

Question 496

What gene do most cancer-causing viruses contain?

Answer 496

An oncogene

Question 497

Why is an oncogene of evolutionary benefit to cancer-causing virus?

Answer 497

Because virus numbers greater from proliferating cells

Question 498

Why does the concept of dominant oncogenes alone not explain cancer cell behaviour?

Answer 498

Because tumour suppressor genes are lost during oncogenesis

Question 499

What was the importance of Knudsen's one/two-hit hypothesis on retinoblastoma?

Answer 499

-for tumour suppressor gene hypothesis -that cancer requires loss of both wild-type alleles -for the basis of inherited predisposition to cancer

Question 500

Which genes are significant in inherited forms of cancer?

Answer 500

tumour suppressor genes, cause uncontrolled proliferation without

Question 501

What is the difference between oncogenes and tumour suppressing genes?

Answer 501

- oncogenes are activating tumour suppressors and are inactivating - oncogenes are dominant and lead to gain of function whilst tumour suppressions are recessive and lead to loss of function - oncogenes need to mute 1 glee to exert effect whilst tumour suppressors need to mutate 2 - oncogenes enhance protein product tumour suppressors reduce it

Question 502

Describe turmeric evolution?

Answer 502

- Successive rounds of random inherited change and natural selection underpins tumour progression - Cancer cells are genetically unstable, genetic instability contributes to tumorigenic evolution - Increased cell divison AND decreased apoptosis contribute to tumorigenesis Ability to undergo apoptosis compromised for cancer cells, both increased rate of proliferation due to gain of oncotic genes and tumor suppressor genes

Question 503

What are the causes of genetic instability?

Answer 503

Defects in: -DNA repair pathways -Correction mechanisms for DNA replication errors -Correction mechanisms for DNA segregation errors (Without correction methods copying occurs, no editing, causes genetic instability Errors that occur in mitosis cause genetic instability)

Question 504

Normal cells respond to cellular stresses through one critical signalling pathway, what is the gene included in this?

Answer 504

P53 P53 is a gene important in ……DNA damage , etc P53 has an escalating capability Can arrest cell cycle (for example can stop damaged DNA from replication, gives cells time to repair) If they can't be repaired, they senescence, cell take out Then cell is killed by apoptosis

Question 505

What gene is mutated in almost all types of cancer?

Answer 505

P53 Mutations in p53 and associated pathways disrupt intrinsic apoptosis

Question 506

What kind of gene is P53?

Answer 506

Cell cycle checkpoint gene Checkpoints monitor the cell cycle, stop progression if there is a problem Checkpoint mechanisms can’t operate without p53 Cell cycle will continue regardless of DNA damage Will replicate with variable genetic status

Question 507

What does cell-cycle checkpoint one loss do?

Answer 507

Contributes to genetic instability Checkpoints mutated in cancer

Question 508

What is the significance of Rb in cancer?

Answer 508

Restriction point – where cells decide if environment is appropriate for proliferation Rb is a stopping device Stops cell cycle In checkpoints there is mechanism for stopping Rb Cells without Rb will not stop Lots of tumors in those with mutated RB

Question 509

What is the difference between benign cancer and malignant cancer

Answer 509

Benign tumour cells remain within local tissue structure (defined by basal lamina) Malignant tumour cells destroy integrity of tissue and break through tissue limits (invasiveness

Question 510

What does malignant invasiveness (tumour cells destroying tissue) give rise to?

Answer 510

Metastasis | -The development of secondary malignant growths at a distance from a primary site of cancer.

Question 511

Explain the TNM staging system?

Answer 511

T refers to the size and extent of the primary tumour. N refers to the number of nearby lymph nodes that have cancer M refers to whether the cancer has metastasized

Question 512

What is tumour staging?

Answer 512

Stage refers to the extent of your cancer, such as how large the tumor is, and if it has spread

Question 513

What is the benefit of staging tumours?

Answer 513

Tells you: - Where the tumor is located in the body - The cell type (such as adenocarcinoma or squamous cell carcinoma ) - The size of the tumor - Whether the cancer has spread to nearby lymph nodes - Whether the cancer has spread to a different part of the body - Tumour grade, which refers to how abnormal the cancer cells look and how likely the tumor is to grow and spread

Question 514

Explain the measure of T in the TNM staging system?

Answer 514

TX: Main tumour cannot be measured T0: Main tumour cannot be found T1, T2, T3, T4: Refers to the size and/or extent of the main tumour. The higher the number after the T, the larger the tumour or the more it has grown into nearby tissues.

Question 515

What qualities should a cancer test have?

Answer 515

- cheap - minimally invasive - accurate - sensitive (low limit of detection)

Question 515

What qualities should a cancer test have?

Answer 515

- cheap - minimally invasive - accurate - sensitive (low limit of detection)

Question 516

Why is secondary screening important?

Answer 516

Secondary screening is increasingly important for decisions regarding treatment e.g A subset of breast cancers are dependent on over-expression of a specific growth factor receptor (Her2)

Question 517

Why is secondary screening important?

Answer 517

Secondary screening is increasingly important for decisions regarding treatment e.g A subset of breast cancers are dependent on over-expression of a specific growth factor receptor (Her2)

Question 518

What can be used specifically to treat breast tumours that over-express Her2

Answer 518

Hercepin - monoclonal antibody binds to erbB2 receptors - causes cell cycle arrest (G1)

Question 519

What are gas proteins?

Answer 519

proto-oncogenes (important in normal cell growth) that are frequently mutated in human cancers

Question 520

What is the importance of ras in growth factor-induced growth

Answer 520

Ras, a small GTP-binding protein, is an important component of the signal transduction pathway used by growth factors to initiate cell growth and differentiation. Cell activation with growth factors such as epidermal growth factor (EGF) induces Ras to move from an inactive GDP-bound state to an active GTP-bound state. Mutant RAS protein is permanently stuck in the “on” position, constantly activating downstream signaling pathways and promoting growth signals