Molecular And Cell Biology Flashcards
What is Ferritin?
A protein which stores, transports and releases iron.
What is Porin?
A protein which sits in the outer bacterial membrane - allows diffusion of certain molecules.
What is a protein’s secondary structure?
Has hydrogen bonding.
Alpha helix - h bonds between amino and carboxyl groups (4 residues apart)
Beta sheet - h bonds between different strands.
What is a protein’s tertiary structure?
Thermodynamically stable - 3D.
Determined by non-covalent interactions.
Where do amino acids go in a tertiary structure?
Polar residues end up on the outside - so can interact with polar water molecules.
Non - polar fold in centre.
What is a disulphide bridge?
Interaction between sulphur atoms in cysteine.
What is a protein domain?
Some proteins fold into different domains - separated by flexible regions.
They carry out a specific function.
What is a protein’s quaternary structure?
Formed of subunits.
2 = dimer, 3 = trimer, 4 = tetramer.
What is methylation?
Post-translational modification
Adds on a -CH3 group.
E.G. on histones to control genome expression.
What is glycosylation?
Post-translational modification
Adds on sugars.
What is ubiquitination?
Post-translational modification.
Adds a 76aa polypeptide to mark protein for degradation.
What is phosphorylation?
Reversible
Adds PO3 - uses kinase enzymes.
Regulates enzyme function.
What is protein targeting?
Some proteins contain signals or localisation sequences to show where they need to go.
Some are targeted to cell membrane by the secretory pathway (become channel proteins ect).
What are anchor membrane proteins?
Anchored to membrane by additional hydrophobic groups added on - allows them to be be removed from membrane.
E.g. Ras
What is a microtubule?
Made of alpha and beta tubulin
Is there rotation around peptide bonds?
NO
What is a short chain of amino acids called?
Peptide
What is an unfolded protein called?
Denatured then turns native (folded)
What are the Mendelian laws of inheritance?
Segregation: genes come in pairs, one is passed on to offspring.
Independent assortment: genes are passed on separately from eachother.
Dominance: the dominant allele will be expressed.
What was Sutton and Boveri’s chromosome theory of inheritance?
Observed meiosis in grasshoppers and worms (as chromosomes are large and few) - noticed that destroying chromosomes stopped normal embryo development, consistent with mendel’s law
- Chromosomes are required for embryonic development.
- Chromosomes carry ‘Mendel’s factors.
- Chromosomes are linear structures with genes along them.
What is streptococcus pneumoniae?
Causes pneumonia in humans and mice - only some strains.
S strain - smooth, polysaccharide coats - pathogenic.
R strain - rough - no coat - not pathogenic.
The coat forms a capsule which protects strains.
What did Griffith do to the streptococcus?
Heated S strain - no infection
Heated S strain and R strain - infection
This due to R cells transforming as some material from s –> r.
This is the transforming principle.
What did Avery, Macleod and McCarthy do?
No one knows what was passed to R cells.
They destroyed different parts to see what was causing it.
They found it was small pieces of DNA which coded for the capsule.
What is a bacteriaphage?
A category of viruses which require bacterium to be a host cell.
What is bacteriophage T2?
The host is Escherichia coli.
It destroys the chromosomes and replicates its genone.
What experiment did Hershey & Chase do which showed how bacteriophages reproduce?
1) label bacteriophage DNA or protein with radioactive isotope
2) Infect a bacteria (only DNA enters)
3) Separate phage ghosts and bacteria (use blender and centrifuge)
4) test for radioactivity
How did they label bacteriophage with radioactivity?
32P and 35S are unstable isotopes - can be detected by Geiger counter.
Growing the bacteriophage in media with them will produce radioactively labelled protein/DNA.
What did the bacteriosome experiment show?
Supernatant had phage ghosts, pellet had bacteria. They were tested for radioactivity - It showed that DNA was injected, not proteins.
What are the purines and pyrimidines?
Purines - A,G (2 carbon rings)
Pyrimidines - C,T,U (1 carbon rings)
What is a nucleoside?
Sugar + base
What did Chargaff do?
Used chromatography to seperate and isolate nucleobases.
Chargaff’s rules
%A = %T, %C = %G
%AT does not = %GC
What are the main features of Crick and Watson’s model?
A-T and G-C hydrogen bonded pairs
Antiparallel
Right handed double helix
Major (big gap) and minor grooves (little gap)
One helical turn every 10.5 bp
How many hydrogen bonds between AT and CG?
AT = 2
CG = 3
5’ to 3’ polarity
What maintains the DNA width?
The binding of one purine and one pyrimidine
What is one complete turn of DNA?
3.4 nm, 10.5 bp
What is a centromere?
Specialised region where microtubules attach - doesn’t have to be in the centre
What is a telomere?
Repetitive DNA at end of chromosomes
Protect the ends of chromosomes
What is the prokaryotic genome?
Single, circle chromosome
Plasmids also found
Passed between cells by conjugation
Few million bp
What are DNA-binding proteins?
Have domains which can regulate gene expression, cut DNA and protect DNA
e.g. restriction endonucleases, transcription regulators, histones
What is a transcriptional regulator?
Proteins which bind to reg sequences near promotors to stimulate or block transcription. Bend DNA in favourable or unfavourable ways
e.g. lac operon - lac repressor binds to DNA to block transcription
What is a restriction endonuclease?
Enzyme which cuts DNA at specific sequences.
Restricts action of viruses.
Bacteria is protected as methylated
What are histones?
Proteins which chromatin is wrapped around.
What are the bases and nucleosides in RNA?
Uracil - uridine
Adenine - adenosine
Guanine - guanosine
Cytosine - cytidine
What are stem-loop structure?
Short helices in RNA caused by intramolecular base-pairing.
Secondary structural elements.
e.g. tRNA
What do most interactions in RNA occur in?
Minor groove - major too narrow
What is non-canonical base-pairing?
G-U, A-C - wobble base pair
These can stabilise RNA
Why is deoxyribose more stable than ribose?
Lacks a OH on second carbon
The OH makes ribose more reactive and prone to hydrolysis
What is the secondary and tertiary structure of RNA?
Secondary - 2D map defined through intramolecular base-pairing.
Tertiary - interactions that connect regions separated in secondary structure - can be canonical.
What is the A minor motif?
Two adjacent A bases interacting with the edge of a G-C base pair.
How is RNA made?
From RNA polymerases - transcription.
RNAP active site contains a short RNA/DNA heteroduplex
Where does RNA polymerase start?
Promotor regions until reaches terminator region.
What is the E.coli RNA polymerase core enzyme?
Protein complex containing 5 subunits:
2 alpha - binds transcription factors
2 beta - catalytic
w - assembly and stability
What are sigma factors?
Provide specificity to RNAP for the gene promotor.
In prokaryotes
Released from RNAP
How many RNA polymerases in eukaryotic cells?
I = rRNA
II = mRNA, noncoding RNA
III = tRNA, 5s RNA
Have conserved core structure
What is a gTF?
General transcription factor
Required to assemble RNAP II onto promotors in eukaryotic cells.
A preinitiation complex involves a multi-step pathway.
e.g. TFIIA, TFIIB
How did Wilkins and Franklin see DNA?
X-ray crystallography - saw x pattern (helix), regular pattern, distance between spots (one turn) = 3.5 nm
What did Meselson and Stahl do?
Used nitrogen isotopes to experiment semi-conservative DNA.
14 and 15 - nitrogen
1) grew the bacteria in media to make heavy (15) and light (14) DNA
2) separate by ultracentrifugation
3) mix with caesium chloride
4) look at DNA using UV light
5) saw different bands after every generation
1st - 1 band
2nd - 2 bands
3/4/5 - 2 bands but hybrid getting lighter and lighter
How many replication origins?
1 in E.coli
Tens of thousand in humans - bidirectional replication forks
What does DNA polymerase do?
Adds nucleotides one at a time 5’-3’ using template strand
What does primase and ligase do in DNA replication?
Primase generates the primer
Ligase joins the new DNA together (loose ends into a single strand)
What do topoisomerase and helicase do in DNA replication?
Topoisomerase relieves pressure from overwinding by breaking and resealing DNA.
Helicase breaks H bonds between two strands
What is the single-strand binding protein?
Prevents the two DNA strands from reannealing in DNA replication
What are the leading and lagging strands?
Leading strand - DNA points towards replication fork - continuous
Lagging - DNA points away so must be discontinued and primed multiple times
BOTH 5’-3’
What are okazaki fragments?
Pieces of DNA - they are stuck together to make lagging strand
Why is there erosion at the end of chromosomes?
Primer is removed - leaves a gap so lagging strand is incomplete - telomeres solve this - telomerase can replenish telomeres
How are RNAs resolved?
Large (rRNA) - in agarose gels
Small - acrylamide gels
mRNA is not clearly seen
What are the major cellular RNAs?
mRNA - 5%
rRNA - 75%
tRNA - 10%
How is mRNA processed?
- capped at 5’ end
- pre-mRNA splicing
- 3’ end processed (cleavage and polyadenylation - adding a stretch of adenine nucleotides)
- happens in nucleus
- the cap and poly(A) protects and promotes translation
What is the m7G cap?
A guanosine nucleotide is added to the 5’ end of RNA pol II transcripts - linked by 5’5’ triphosphate linkage.
Then methylated
What is polycistronic?
Where an mRNA can code for multiple proteins - in prokaryotic cells
What are split genes?
Genes are interrupted by introns
Prokaryotic cells DO NOT HAVE THIS
How is pre-mRNA splicing accurate?
Introns and exons have splice site sequences
5’ - GU (start of intron)
3’ AG (end of intron)
Introns also have a branchpoint ‘A’
What causes splicing?
Carried out by a ribonucleoprotein (RNP) called spliceosome
These are made from smaller RNA/proteins called snurps
What are the steps in splicing?
Involves 2 transesterification steps -
1) 5’ exon removed and the intron forms a lariat structure involving branchpoint adenosine
2) exons joined and intron lariat released
Can some RNA’s undergo self splicing?
Yes!
Nuclear splicing is said to have evolved from this
Enzymes with RNA catalytic subunit - ribozyme
What does understanding the genetic code allow us to do?
- infer protein sequences from DNA and its sequence
- infer protein function by comparison
- design tools to study protein function e.g. drugs
How are mRNA codons recognized?
By base pairing with anticodons within cognate tRNAs - the tRNAs are charged/aminoacylated (connected to appropriate amino acid) - mediated by aminoacyl-tRNA synthetases
How does the amino acid on tRNA bind to the chain?
Peptidyl group and aminoacyl groups bind - peptide bond
What’s the name for ‘all nucleotides have a genetic meaning’?
Nonpunctuated
Some have same meaning - degenerate
How was the genetic code cracked?
Cell extracts were programmed to make proteins using artificial RNAs made with polynucleotide phosphorylase.
e.g. poly(u) RNA made phenylalanine
How many codons does the genetic code have?
64 - 61 are sense = code for amino acids
- 3 are stop codons - UAA, UAG, UGA
The start codon is AUG - methionine
Met and Trp have unique codons - others degenerate
What are synonymous codons?
Code for same amino acid - usually differ by 3rd nucleotide
Some recognised by isoacceptor tRNA’s - charged with same amino acids
Others can be recognised by the same tRNA - wobble base-pairing
What nucleotide do many tRNA’s have?
Inosine - 1st position on anticodon
Can pair with U,A,C
G can pair with C or U
What is the tRNAs secondary structure?
Cloverleaf - 5’ and 3’ drawn together
Amino acid is bound to 3’ hydroxyl on A nucleotide
All tRNA have 3’ terminal CCA
Modification of 1st position of anticodon allows wobble
What is coaxial stacking?
Increases thermodynamic ability in tRNA
Anticodon on D arm, acceptor on TYC arm
Base pairing between the arms
How is the tRNA charged?
Mediated by aminoacyl-tRNA synthetases - needs ATP
20 different ones - causes ester link between carboxylic group of AA and 3’ hydroxyl group
What is the ribosome structure?
Large RNP particle - has two unequal subunits
Codon/anticodon occus on small subunit
Peptide bonding occurs on large subunit
There are 3 tRNA binding sites (A,P,E)
Has a peptidyltransferase centre (PTC) - RNA rich - makes peptide bonds - RNA catalysed
How are ribosomes synthesised?
rRNA transcription and early steps in nucleoli
Later steps in nucleosomes and cytoplasm
What happens during translation elongation?
2 tRNAs are bound at a given time, either at A & P, or P & E.
Contains cycles of:
- aminoacyl-tRNA binding
- peptide bond formation
- translocation of the ribosome along mRNA
When aminoacyl-tRNA binds to A - tRNA is released from E
What do A, P and E mean in ribosomes?
P = peptidyl site
A = aminoacyl site
E = exit site
How are GTPases used in translation elongation?
The aminoacyl-tRNA brought to ribosome by the elongation factor EF1A (EF-Tu in prokaryotes)
Translocation needs EF2 (EFG in prokaryotes)
Both are GTPases - 2 GTP molecules are hydrolysed per cycle
How does the tRNA go to the start codon?
EF1a brings Methionyl-tRNAs to bind to AUG codons and associate with eukaryotic initiation factor 2, eIF2 (IF2 in prokaryotes)
What is the Shine-Delgarno sequence?
Allows the start codon to start at P site - in prokaryotes - near the start codon - 3’ end of 16s rRNA
How is translation initiated in eukaryotes?
tRNAmet (bound to eIF2) and 5’ mRNA bind through interaction with cap-binding complex.
The preinitiation complex scans along mRNA - finds AUG - kozak sequence
The large subunit is then recruited
How is translation terminated?
Eukaryotic release factor 1 (eRF1) or RF1/2 in prokaryotes trigger peptide hydrolysis .
eRF3 (GTPase) releases peptide by releasing RF1/2 from ribosome
What are housekeeping genes?
Genes that are constitutively expressed
What mutations can effect regulatory elements?
cis - mutation within the same gene - DNA sequences that effect gene regulation
trans - in a different gene - protein or RNA factors that regulate the expression of target gene
What stage are genes mostly regulated?
Transcriptional
What are transcription factors?
Can activate (activators) - positive control - at weak promotors
Can repress (repressors) - negative control
Activators in E.coli interact with a subunit of RNA polymerase - promote binding
How are transcription factors modulated?
Inducers - bind to TF and stimulate activators or inhibit repressors
Corepressors - bind to TF and stimulate repressors or inhibit activators
What is the lac operon?
Codes for 3 genes - lacZ, lacY and lacA
Controlled by transcription repressor gene - lacI
LacI - binds to operator (lacO) and blocks RNA polymerase
Lactose is the inducer - inhibit repressor
Negative - inducible
What is CAP in lac operon?
lac operon also regulated by activator - catabolite activator protein
Binds to lac promotor when associated with cAMP (inducer) - stims transcription
cAMP is inhibited by glucose (ideal energy source)
Lac operon - expressed by absence of glucose and presence of lactose
How can RNA processing control gene expression?
Pre-mRNA can occur in different patterns - different proteins
How does translation control gene expression?
Translation is downregulated by integrated stress response
Can also be regulated by specific mechanisms e.g. Ferritin expression is responsive to Fe2+
How does post-translational modifications regulate protein function?
Most commonly - phosphorylation of serine, threonine or tyrosine by kinases
How is translation inhibited by eIF2a?
eIF2 hydrolyses GTP to GDP - regeneration requires eIF2B but eIF2a is phosphorylated by integrated stress response and binds very tightly to it so it cannot be recycled
What are the basic steps of molecular cloning?
1) insert DNA into a vector (usually a plasmid) - makes recombinant DNA
2) transfer to host
3) replicate
What features must a vector have?
1) origin of replication - replication in host
2) selectable marker - survival of host cells e.g. antibiotic resistant gene
3) multiple cloning site - where we insert gene - has restriction enzyme sites
How do we cut up DNA?
Use restriction enzymes - recognise short specific sequence
There are 3/4 types:
- types I and III cleave DNA at random places far from recognition sequence
- type IV cleave modified DNA
- type II cut DNA at a specific place
Name them: Species, Strain, Enzyme e.g. EcoRV
What are type II restriction enzymes?
Most commonly protein homodimers
DNA sequence usually palindromic
Recognise specific DNA sequence
Can generate overhangs or blunt edges - overhangs are compatible
How do type II restriction enzymes work?
1) initial binding is non-specific
2) moves along until finds specific site
3) this binding causes structural changes
4) catalysis requires Mg2+
5) generates 5’ phosphate and 3’ OH ends
What is the ligation reaction?
Sticky ends have to interact - DNA ligase catalyses new phosphodiester bond
What problems may molecular cloning have?
- insert doesn’t join vector
- little DNA
- DNA may be mixed into lots of other molecules
How do we modify the DNA ends in molecular cloning?
Removal/addition of 5’ phosphate - need to form phosphodiester so needs to be there
Adding - T4 polynucleotide kinase
Removing - Calf Intestinal Phosphatase (CIP) - this can prevent self-ligation
How can we make blunt ends?
Fill in 5’ overhang or remove 3’ overhang - T4 DNA polymerase or DNA polymerase I
Remove 5’ overhang - Mung bean nuclease
How do we make host cell take in recombinant DNA?
1) electroporation = brief high voltage
2) chemical trasformation - heat shock - causes membrane changes that allow DNA uptake
- not that efficient - use antibiotics to find cells with the DNA
What is PCR?
DNA replication in a tube - DNA doubles each cycle
can solve cloning problems - not having enough DNA and DNA might be mixed with other DNA
What are the stages of PCR?
1) denaturing - DNA dissociates into single strands at high temperatures (95)
2) primer annealing - primers bind to complementary sequence (55-65)
3) primer extension - DNA polymerase synthesises new strands from 3’ end of primer (68-72)
Which DNA polymerase is used in PCR?
Taq - thermostable and has high processivity and extension rate (2-4 kb per minute) however - low accuracy and adds adenine overhang - no proofreading
Pfu - better thermostability, slower extension rate (1kb per minutew), products are blunt ended, more accurate
What features do PCR primers have?
- has to be minimum 17 base pairs
- be specific to template
- come in pairs
- appropriate melting temp
What do we need for PCR?
Template
DNA polymerase
Primers
Deoxyribonucleoside triphosphate (dNTPs)
Buffer
Thermocycler
What is the melting temperature (Tm) in PCR?
Temperature that primer dissociates from DNA - 60/64 degrees
- determines what the annealing temp should be (5 less than Tm)
To work out: add 4 degrees for G/C or 2 for A/T
If too low - primers may bind to other parts of sequence
If too high - may not bind at all
Can we put a PCR product into a vector?
Yes! - there’s no phosphate at the end - add T4 PNK, taq has overhang which needs to be removed - T4 DNA pol
We can add sequences at 5’ end which is complementary to restriction enzymes.
What is reverse transcription PCR?
RNA is reverse transcribed into DNA (cDNA - copy/complementary) - this is then amplified with PCR
How do we make cDNA?
reverse transcriptase synthesises the first strand of cDNA:
1) poly(dT) primers bind poly(A) tail of mRNA
2) mRNA removed
This makes first strand
1) synthesised by klenow fragment of DNA polymerase I
2) hairpin formed by RT acts as primer
3) the ssDNA loop can be digested by nuclease
This makes the second
Then do normal PCR
What is quantitative PCR?
PCR but we can see how much DNA at any time
PCR is exponential at the beginning - until things run out
We can measure product by:
- fluorescent dye - SYBR green fluoresces when binds to dsDNA - they are proportional
- fluorescent probe - sequence specific - is bound to DNA until replaced by strand - then fluoresence - we can use several
What is Ct?
Cycle threshold - where fluorescence exceeds background levels
Difference in Ct = relative measure of what sample had most sample. we can do 2^-difference to find difference
Lower CT = more template at start
We can also minus test from sample for A , then B then minus A and B then do 2^-this number
How do we remove our DNA from bacteria?
Mini prep: Grow lots of bacteria - then break them open - plasmids are small - we will have purified recombinant DNA
How do we analyse recombinant DNA?
We can use restriction enzymes to digest the DNA and see if we get the correct sized fragments
We can also use PCR - see if we get the correct product
We can use electrophoresis to separate fragments - add dye (midori green/ethidium bromide)
How can we use restriction sites to analyse recombinant DNA?
If we add restriction sites to the gene and vector, but have different enzymes that will effect them, we can see different patterns if the DNA is not present/inserted wrong
- restriction fragment length polymorphism - been mutated or gotten longer due to disease - won’t bind
How can we use PCR to analyse recombinant DNA?
Use 2 primers: 1 for DNA, 1 for vector
If we get PCR products - the DNA and vector are correctly combined, no product - no DNA or incorrect DNA
- allows us to identify diseases, cancer ect. & genetic fingerprinting
How does Sanger sequencing work?
Allows us to see that there’s no PCR mutations
1) DNA is amplified using one primer
2) ddNTP (dideoxynucleoside triphosphate) is added - stops sequencing as there’s a missing OH (there’s also normal dNTP)
3) If we add one type e.g. ddCTP then we know that’s the last nucleoside
4) we can then use electrophoresis to see how big the fragments are
5) repeat with ddATP, ddGTP and ddTTP
What is modern sequencing?
One reaction has all ddNTP - all have different fluorescent labels - use capillary gel electrophoresis which is read by computer
What are the components of modern sequencing?
- A taq DNA polymerase
- dNTPs
- ddNTPs
- Buffer
- Template DNA
- Primer
What methods can we use to analyse RNA expression?
RT-PCR - we can use different isoforms of mRNA and look at the different types.
Hybridisation-based technique - can use RNA to make a probe (DNA and RNA together)
To see RNA expression we can use northern blots and microarray
To see localisation use fluorescence in situ hybridisation
When we read DNA, which way do we read?
5’ to 3’ so if something was complimentary - make sure we reverse
What is northern blot?
1) RNA is separated by electrophoresis (by size)
2) transfer RNA to membrane to allow for blotting
3) add a probe (radioactive) then wash membrane - unprobed will wash away
This is good for comparing mutated genes ect. - can only use one molecule at a time