Ch 7 - 13 Flashcards
(113 cards)
1
Q
RNA
A
RNA is largely single-stranded
RNA can fold into specific three-dimensional structures that are determined by its sequence of nucleotides that base pair via Watson-Crick conventional and “nonconventional” base-pair interactions (e.g. A-G)
2
Q
template vs coding strands
A
Transcription of a gene produces an RNA complementary to the template strand of DNA
The coding strand (nontemplate strand) is equivalent to the RNA product
3
Q
RNA polymerase
A
an enzyme that transcribes DNA into RNA
unwinds the DNA and adds ribonucleotides one-by-one to the RNA chain, using the template DNA strand
As the polymerase moves along the DNA template, it displaces the newly formed RNA, allowing the two strands of DNA behind the polymerase to rewind. Therefore, a short region of hybrid DNA/RNA helix (approximately 9 nucleotides long) forms only transiently
Many molecules of RNA polymerase can simultaneously transcribe the same gene
4
Q
What is the function of these different types of RNA produced in cells?
messenger RNAs (mRNAs)
ribosomal RNAs (rRNAs)
microRNAs (miRNAs)
transfer RNAs (tRNAs)
other noncoding RNAs
A
mRNAs: code for proteins
rRNAs: form the core of the ribosome’s structure and catalyze protein synthesis
miRNAs: regulate gene expression
tRNAs: serve as adaptors between mRNA and amino acids during protein synthesis
other noncoding RNAs: used in RNA splicing, gene regulation, telomere maintenance, and many other processes
5
Q
What is are the steps of bacteria gene transcription?
A
- Bacterial RNA polymerase contains a subunit called sigma factor that recognizes the promoter of a gene. The polarity of the promoter orients the polymerase and determines which DNA strand is transcribed.
- Once transcription has begun, sigma factor is released, and the polymerase moves forward and continues synthesizing the RNA
- elongation continues until the polymerase encounters the terminator sequence
- After transcribing this sequence into RNA, the enzyme halts and releases both the DNA template and the newly made RNA transcript. Note that the regions transcribed into RNA contain the terminator but not the promoter nucleotide sequences.
- The polymerase then reassociates with a free sigma factor and searches for another promoter to begin the process again
6
Q
True or False.
All genes are transcribed using the same DNA strand as a template.
A
False.
On an individual chromosome, some genes are transcribed using one DNA strand as a template, and others are transcribed from the other DNA strand.
Which strand will serve as the template is determined by the polarity of the promoter sequences at the beginning of each gene.
RNA polymerase always moves in the 3’ to 5’ direction with respect to the template DNA strand
7
Q
What are the genes transcribed by each of the RNA polymerases below?
RNA polymerase I
RNA polymerase II
RNA polymerase III
A
RNA polymerase I: most rRNA genes
RNA polymerase II: all protein-coding genes, miRNA genes, plus genes for other non-coding RNAs (e.g., spliceosomes)
RNA polymerase III: tRNA genes, 55 rRNA gene, genes for many other small RNAs
8
Q
Describe the beginning of a eukaryotic transcription
A
- A subunit of a general transcription factor (TFIID) - the TATA-binding protein - binds to the DNA sequence in the promoter called the TATA box, bending the DNA double helix
- The binding of TFIID enables the adjacent binding of TFIIB
- This allows the rest of the general transcription factors and RNA polymerase II to assemble at the promoter (at specific sequences)
- TFIIH pries apart the double helix at the transcription start point using ATP, exposing the template strand of the gene
- TFIIH also phosphorylates the long polypeptide “tail” on the RNA pol to release it from the general transcription factors and begin transcription
- once the polymerase moves away from the promoter, most of the general transcription factors are released from the DNA except TFIID, which remains bound through multiple rounds of transcription initiation
9
Q
nucleolus
A
where ribosomal RNAs are synthesized and combined with proteins to form ribosomes, which are then exported to the cytosol
one of many “factories” - intracellular condensates - that synthesize and process RNAs within the nucleus. Proteins involved in DNA replication and repair also converge to form functional factories
10
Q
True or False.
RNA-processing occurs after an RNA molecule is fully transcribed.
A
False.
RNA processing - capping (5’), splicing, and polyadenylation (3’) - take place as the RNA is being synthesized. Phosphorylation of the tail of RNA polymerase II allows RNA-processing proteins to assemble there. RNA processing occurs as the RNA is being synthesized; as the RNA molecule emerges from the polymerase.
The phosphates shown here are in addition to the ones required for transcription initiation.
11
Q
Name the key structures of a modified eukaryotic mRNA molecule.
What is the purpose of eukaryotic mRNA modification?
A
5’ cap: guanine nucleotide bearing a methyl group; not on bacteria
3’ poly-A-tail: a few hundred nucleotides long; mRNA trimmed by an enzyme then polyadenylated by the second enzyme; not on bacteria
noncoding sequences: 5’ untranslated region (UTR) and 3’ UTR
modifications increase the stability of the eukaryotic mRNA molecule, facilitate its export from the nucleus to the cytosol, and generally mark the RNA molecule as an mRNA. They are also a way for the protein-synthesis machinery to check that both ends of the mRNA are present and that the message is complete before protein synthesis begins.
12
Q
What is the difference between a eukaryotic and bacterial genes?
A
A bacterial gene consists of a single stretch of an uninterrupted nucleotide sequence that encodes the amino acid sequence of a protein.
The protein-coding sequences of most eukaryotic genes (exons) are interrupted by noncoding sequences (introns)
13
Q
Describe the intron splicing process.
A
special nucleotide sequences in a pre-mRNA transcript signal the beginning and the end of an intron.
snRNPs (RNA-protein complexes) recognize these sequences (5’ splice site and the lariat branch-point site) through complementary base-pairing.
conformational changes in the snRNPs triggered by ATP hydrolysis drive the formation of the spliceosome active site (branch-point cuts the sugar-phosphate backbone of the RNA at the 5’ splice site to form a branched structure)
The free 3’-OH end of the exon sequence reacts with the start of the next exon sequence, covalently joining the two exons together
Once the splicing reactions have occurred, the spliceosome deposits an exon junction complex on the mRNA to mark the splice site as successfully completed
The intron is released as a lariat structure, which is eventually degraded in the nucleus
14
Q
alternative RNA splicing
A
spliceosomes can skip over some exons to produce different mRNAs and thus proteins from the same gene.
Such skipping occurs when the splicing signals at the 5’ end of one intron are paired up with the branch-point and 3’ end of a different intron
exons can be skipped or included but their order cannot be rearranged!
15
Q
How does the nucleus know an mRNA is ready for export?
A
a specialized set of RNA binding proteins mark the 5’ cap and poly-A-tail of a mature mRNA
Once the mRNA is deemed export-ready, a nuclear transport receptor associates with the mRNA and guides it through the nuclear pore
in the cytosol, the mRNA can shed some of these proteins and bind new ones, which act as initiation factors for protein synthesis
16
Q
What are the differences in the transcription and translation of mRNA molecules in a eukaryote vs prokaryote?
A
The transcription of eukaryotes occurs in the nucleus, and the translation occurs in the cytosol. The mRNAs are degraded by RNAses in the cytosol and their nucleotides are reused for transcription.
In prokaryotes, there is no modification of the RNA molecule. Transcription, translation, and degradation take place in the cytosol (because there’s no nucleus). Translation and transcription can occur at the same time.
17
Q
tRNA
A
link amino acids to codons
the single-stranded RNA molecule base-pairs with itself to create the double-helical regions of the molecule; tRNAs contain some unusual bases, which are produced by uracil modification after the tRNA has been synthesized
The anticodon loop contains the sequence of three nucleotides that base-pairs with the codon in mRNA
the amino acid matching the anticodon is attached at the 3’ end of the tRNA
18
Q
aminoacyl-tRNA synthetases
A
there is a different synthetase enzyme for each amino acid
each aminoacyl-tRNA synthetase makes multiple contacts with its tRNA molecule; nucleotides in both the anticodon loop and amino-acid-accepting arm are recognized
ATP is used to covalently attach the amino acid to the appropriate tRNA, a process called charging
19
Q
ribosomes
A
located in the cytoplasm of eukaryotic cells
a large complex of 4 rRNAs and more than 80 small proteins; the RNAs account for most of the mass of the ribosome and give it its overall shape and structure.
formed from a large and small subunit, which only come together after the small subunit has bound an mRNA
Each ribosome has three binding sites for tRNAs (A, P, and E sites); both the large and small subunits are involved in forming the sites; during protein synthesis, only two of these sites are occupied at any one time
20
Q
Describe the translation process
A
- a charged tRNA carrying the amino acid to be added to the polypeptide chain binds to the A site on the ribosome by forming base pairs with the mRNA codon
- the carboxyl end of the polypeptide chain is uncoupled from the tRNA at the P site and joined by a peptide bond to the free amino group of the amino acid linked to the tRNA at the A site
- a shift of the large subunit relative to the small subunit moves the two bound tRNAs into the E and P sites of the large subunit
- the small subunit moves back to its original position relative to the large subunit; this ejects the spent tRNA and resets the ribosome with an empty A site so that the next charged tRNA molecule can bind
21
Q
How is translation initiated?
A
initiation of protein synthesis requires translation initiation factors and a special initiator tRNA (methionine, which is later removed by a specific protease). The initiator tRNA is different from the tRNA that normally carries methionine; it is the only charged tRNA that can bind to the P site in the absence of the large ribosomal subunit
the small ribosomal subunit loaded with the initiator tRNA binds to the 5’ end of an mRNA molecule, which is marked by the 5’ cap
the small ribosomal subunit then scans the mRNA until it encounters the first AUG; when the AUG is recognized by the initiator tRNA, several of the initiation factors dissociate from the small ribosomal subunit to make way for the large ribosomal subunit to bind and complete ribosomal assembly.
22
Q
prokaryotic mRNA
A
encode several different proteins due to operons (genes directing the different steps in a process organized into clusters)
does not have a 5’ cap, but has a triphosphate at its 5’ end
Translation is initiated by prokaryotic ribosomes binding at ribosome-binding sites, which can be located in the interior of an mRNA molecule (allows simultaneous synthesis of different proteins from a single mRNA molecule with each protein made by a different ribosome)
23
Q
how does translation stop?
A
Release factor (not tRNA) binds to the A site bearing a stop codon, which terminates translation. the completed polypeptide is released, and the ribosome dissociates into its two separate subunits.
24
Q
proteosomes
A
in eukaryotes, proteins are broken down by large protein machines called proteasomes (present in both cytosol and nucleus)
proteosome contains a central cylinder formed from proteases whose active sites face into an inner chamber. each end of the cylinder is plugged by stoppers, which bind the proteins destined for degradation and unfold the protein (using ATP), threading them into the inner chamber of the cylinder.
Once the proteins are inside, proteases chop them into short peptides, which are then jettisoned from either end of the proteosome
25
How do proteasomes select which proteins in the cell should be degraded?
proteins in the stopper of a proteasome recognize proteins marked by a polyubiquitin chain
the stopper unfolds the target protein and threads it into the proteasome's central cylinder, which is lined with proteases that chop the protein to pieces.
26
When do translated polypeptide chains become useful to the cell?
a completed polypeptide must fold correctly into its three-dimensional conformation and then bind any required cofactors and protein partners via noncovalent bonding
many proteins also require one or more covalent modifications (phosphorylation or glycosylation) to become active - or to be recruited to specific membranes or organelles
27
True or False.
A neuron and a liver cell share the same genome.
True.
Both cells contain the same genome, but they express different RNAs and proteins
28
What are the various steps at which gene expression in eukaryotic cells can be controlled? Where is the main site of control?
1. transcription of DNA sequence into RNA (main site)
2. RNA processing
3. mRNA transport
4. mRNA degradation
5. translation
6. protein degradation
7. protein activity
29
transcription regulators
proteins that bind to DNA regulatory sequences and act as a switch to control transcription (turn on or off)
many proteins fit into the major groove and form tight associations (hydrogen bonds, ionic bonds, and hydrophobic interactions) with the base pairs in a short stretch of DNA; this particular structural motif (homeodomain) is found in many DNA-binding proteins
many transcription regulators bind as dimers; such dimerization doubles the area of contact with the DNA, thereby greatly increasing the potential strength and specificity of the protein-DNA interaction
30
Trp operon and transcriptional repressors
operons are a cluster of bacterial genes
the entire operon is controlled by a single regulatory DNA sequence called the Trp operator situated within the promoter; the yellow blocks in the promoter represent DNA sequences that bind RNA polymerase
if the concentration of tryptophan inside a bacterium is low, RNA polymerase binds to the promoter and transcribes the five genes of the Trp operon
if the concentration is high, repressor protein becomes active and binds to the operator, where it blocks the binding of RNA polymerase to the promoter
when the concentration of intracellular tryptophan drops, the repressor falls off the DNA, allowing the polymerase to transcribe the operon again
31
transcriptional activators
turn genes on
proteins that work on promoters that are only marginally able to bind and position RNA polymerase on their own
activator proteins bind to a regulatory sequence on the DNA and interact with the RNA polymerase, helping it to initiate transcription
in bacteria, the binding of the activator to DNA is often controlled by the interaction of a metabolite or other small molecule with the activator protein
32
Lac operon and its transcription regulators
* When lactose is absent: Lac repressor binds to Lac operator and shuts off the expression of the operon
* When lactose is present: allolactose binds to the lac repressor, causing it to undergo a conformational change that releases its grip on the operator
* when glucose is absent: cAMP bind to CAP (bacterial activator protein), allowing CAP to bind to DNA
* when glucose is absent and lactose is present: RNA polymerase binds to the promoter and transcribe DNA
33
How can eukaryotic gene activation occur at a distance?
An activator bound to a distant enhancer attracts RNA polymerase and the general transcription factors to the promoter; activators can bind to DNA as dimers and as monomers
Looping of the intervening DNA permits contact between the activator and the transcription initiation complex (mediator) bound to the promoter
The broken stretch of DNA signifies that the segment of DNA between the enhancer and the start of transcription varies in length, sometimes reaching tens of thousands of nucleotide pairs
the TATA box is a DNA recognition sequence for the first general transcription factor that binds to the promoter
34
How is eukaryotic transcription initiated?
eukaryotic transcriptional activators recruit proteins
histone-modifying enzymes: adds acetyl groups to specific histones, which can serve as binding sites for proteins that stimulate transcription initiation
chromatin-remodeling complexes render the DNA packaged in nucleosomes more accessible to other proteins in the cell, including those required for transcription initiation (ex: increased exposure of the TATA box)
35
What prevents a transcription regulator – bond to the control region of one gene – from looping in the wrong direction and inappropriately influencing the transcription of a neighboring gene?
To avoid unwanted cross-talk, the chromosomal DNA of plants and animals is arranged in a series of loops (topological associated domains/TADs) that hold individual genes and their regulatory regions in rough proximity
the loops are formed by specialized proteins that bind to sequences that are then drawn together to form the base of the loop
these loops are much larger than the loops that form between regulatory sequences and promoters
36
combinatorial control
eukaryotic genes are controlled by combinations of transcription regulators
whereas the general transcription factors that assemble at the promoter are the same for all genes transcribed by RNA polymerase, the transcription regulators and the locations of the regulatory sequences are different for different genes
These regulators, along with the chromatin-modifying proteins, histone-modifying enzymes, general transcription factors, and RNA polymerase are assembled at the promoter by the Mediator
The effects of multiple transcription regulators combine to determine the final rate of transcription initiation
37
How can a single transcription regulator coordinate the expression of many different genes?
Different genes containing regulatory DNA sequences that are recognized by the same transcription regulator allow the turning on or off as a coordinated unit
For example, on the left is a series of genes, each with a different activator protein bound to its respective regulatory sequences. However, these bound proteins are not sufficient on their own to activate transcription efficiently
The activated cortisol receptor complex binds to the same regulatory DNA sequence in each gene, completing the combination of transcription regulators required for efficient initiation of transcription. All htre genes are now switched on as a set.
38
How do eukaryotes generate different cell types?
A relatively small number of regulators acting in different combinations can generate many specialized cell types
A small number of transcription regulators can convert one differentiated cell type directly into another (via artificial introduction)
a combination of transcription regulators can induce a differentiated cell to de-differentiate into a pluripotent cell. iPS cells can proliferate indefinitely and can be stimulated by appropriate extracellular signals to differentiate into almost any cell type in the body
39
How can a proliferating cell maintain its identity?
A positive feedback loop can generate cell memory.
A master transcription regulator can activate the transcription of its own gene – as well as other cell-type-specific genes (not shown).
Each time a cell divides, the regulator is distributed to both daughter cells, where it continues to stimulate the positive feedback loop
40
What is DNA methylation and how does it work?
DNA methylation is another way to reinforce cell identity
methylation on cytosine bases that fall next to guanine in the sequence
this modification turns off the affected genes by attracting proteins that bind to methylated cytosines and block gene transcription
DNA methylation patterns are passed on to progeny cells by the action of an enzyme that copies the methylation pattern on the parent DNA strand to the daughter DNA strand as it is synthesized
An enzyme called a maintenance methyltransferase interacts with the hybrid double helices and methylates only those GC sequences that are base-paired with a GC sequence that is already methylated.
41
True or False.
Histone modification is part of a mechanism for inheriting gene expression patterns.
True. Histone modifications may be inherited by daughter chromosomes.
Each daughter chromosome will inherit about half of its parent's collection of modified histones.
Enzymes responsible for these modifications may bind to the parental histones and confer the same modifications to the new histones nearby.
This helps re-establish the pattern of chromatin structure found in the parent chromosome
42
What are the two ways bacteria can regulate translation of its mRNA
1. sequence-specific translation repressor protein keeps the ribosome from binding to the ribosome-binding sequence in the mRNA
* ribosomal proteins made in excess inhibit translation of its own mRNA
* as new ribosomes are assembled, the levels of the free protein decrease, allowing the mRNA to again be translated and the ribosomal protein to be produced
2. thermosensor RNA sequence: at warmer temperatures inside a host, base pairs within the thermosensor come apart, exposing the ribosome-binding sequence, so the necessary protein is made
43
MicroRNAs
targets complementary mRNAs for destruction
miRNA assembles with a set of proteins into a complex called RISC
RISC searches for mRNAs that have a nucleotide sequence complementary to its bound miRNA
extensive match: mRNA rapidly degraded by nuclease in RISC
less extensive match: mRNA transferred to an area of the cytoplasm where other nucleases destroy it
44
small interfering RNAs (siRNAs)
protect cells from infections during the process of RNA interference (RNAi)
foreign RNA molecules are usually long, double-stranded
Dicer cleaves the foreign RNA to make siRNAs, which are incorporated into RISCs
RISC discard one strand of the duplex and use the other strand to locate and destroy foreign RNAs that contain a complementary sequence
45
RNAi and transcriptional silencing
single-stranded siRNA is incorporated into a RITS complex
the RITS searches for complementary RNA sequences as they emerge from a transcribing RNA polymerase
the binding of RITS attracts proteins that promote histone methylation and heterochromatin formation, causing transcriptional repression
46
How do long noncoding RNAs regulate mammalian gene activity?
long noncoding RNAs can serve as scaffolds, bringing together proteins that function in the same cell process
by engaging in complementary base-pairing with other RNA molecules, these long noncoding RNAs can localize proteins to specific sequences in RNA or DNA molecules
47
What are the different mechanisms that alter a genome?
1. mutation within a gene
2. mutation in regulatory DNA
3. gene duplication and divergence
4. exon shuffling
5. transposition
6. horizontal transfer
48
Germ-line cells vs somatic cells
germ-line cells
* genetic information is propagated into the next generation
* gametes that contain only half the number of chromosomes
* can pass on mutations to the next generation
somatic cells
* form the body of the organism
* do not contribute their DNA to the next generation
* cannot pass on mutations to the next generation; responsible for most human cancers
49
Give two examples of changes in regulatory DNA sequences
a point mutation in the regulatory DNA sequence of the lactase gene
When the product of gene 1 turns on different genes because the regulatory DNA sequences controlling the expression of the two genes are different; a collection of such regulatory changes can have profound effects on an organism's development (responsible for differences in species)
50
What can cause gene duplication?
Give an example of duplication and divergence
caused by unequal crossovers between short repeated DNA sequences in adjacent homologous chromosomes (homologous recombination)
* when crossing-over occurs unequally, one chromosome will get two copies of the gene, while the other will get non.
* if this process occurs in the germline, some progeny will inherit the long chromosome, while the others will inherit the short one
The repeated round of duplication and mutation generated the globin gene family in humans. The ancestral globin gene encoded a single-chain globin molecule
51
whole-genome duplication
can happen if cell division fails to occur following a round of genome replication in the germline of an individual
common in plants; make them easier to cultivate, or made their fruits larger, more flavorful, or devoid of seeds
52
exon shuffling
an exon from one gene can be added to another, creating new genes (evolution of new proteins)
occurs by homologous recombination within the introns
nearly all proteins encoded by the human genome arose from the duplication and shuffling of exons
53
mobile genetic elements
DNA sequences that can move from one chromosomal location to another
often disrupt the function or alter the regulation of existing genes; sometimes they create new genes through fusions between mobile sequences and segments of existing genes (coding ore regulatory sequences)
a major source of developmental changes:
* mutations due to mobile genetic element can induce dramatic alterations in the body plan of an organism (drosophila grow legs instead of antennae)
* produce modern corn
54
horizontal gene transfer
genomes exchanged between individuals of different species
rare among eukaryotes, common among bacteria by the process of conjugation
55
What is the idfferences in nucleotide sequence between a
a) human and orangutans
b) human and chimpanzee?
a) 3%
b) 1.2%
56
True or False.
The coding sequence of exons is much more conserved than that of introns across all species.
True
57
DNA-only transposons
most common mobile genetic elements in bacteria
they move by two types of mechanism; a particular type of transposon moves by only one of these mechanisms
1. cut-and-paste transposition: the element is cut out of the donor DNA and inserted into the target DNA, leaving behind a broken donor, which is subsequently repaired
2. replicative transposition: the element is copied by DNA replication; the donor remains unchanged and the target receives a copy of the mobile genetic element
the donor and target DNAs can be part of the same DNA molecule or reside on different DNA molecules
transposons contain the components they need for transposition - transposase and DNA sequences recognized by the transposase; some carry additional genes that encode enzymes that inactivate anitbiotics
58
How can mobile genetic elements move exons from one gene to another?
When two mobile genetic elements of the same type happen to insert near each other in a chromosome, the transposition mechanism occasionally recognizes the ends of two different elements (instead of the two ends of the same element)
The chromosomal DNA that lies between the mobile genetic elements gets excised and moved to a new site
this can generate new genes or alter gene regulation
59
retrotransposons
mobile genetic elements unique to eukaryotes (L1 element and Alu sequence)
retrotransposons are first transcribed into an RNA intermediate
then, a double-stranded DNA copy of this RNA is synthesized by reverse transcriptase
this DNA copy is then inserted into the target location, which can be on the same or different DNA molecule
the donor retrotransposon remains at its original location, so each time it transposes, it duplicates itself
60
describe the processes of infection by a retrovirus
the retrovirus genome consists of an RNA molecule that is packaged with a reverse transcriptase inside a protein coat, which is surrounded by a lipid bilayer that contains virus-encoded envelope protein
the reverse transcriptase makes a single-stranded DNA copy of the viral RNA and then a second DNA strand, generating a double-stranded DNA copy of the RNA genome
this DNA double helix is integrated into a host chromosome
then the new viral RNA molecules are made by the host-cell RNA polymerase
61
single-nucleotide polymorphisms (SNPs)
points in the genome that differ by a single nucleotide pair between one portion of the population and another
by convention, to count as a polymorphism, a genetic difference must be present in at least 1% of the total population of the species
most SNPs are silent; they fall within noncritical regions of the genome
when comparing any two humans, there's about one SNP per every 1000 nucleotide pairs
62
restriction enzymes
obtained from bacteria; cut both strands of the DNA double helix at specific nucleotide sequences
target sequences are often palindromic
some enzymes cut straight across, leaving two blunt ends; others cut staggered, generating "sticky ends"
63
recombinant DNA
two DNA fragments produced by restriction enzymes joined together by ligase; can be from the same restriction enzyme or different enzyme
the fragments can be from different cells, tissues, or even different organism
the key step to DNA cloning
64
plasmids
vectors used to carry the DNA that is to be cloned
circular, double-stranded DNA molecules
contains its own replication origin, which enables its to replicate in a bacterial cell independently of the bacterial chromosome
has cleavage sites for common restriction enzymes
the recombinant plasmid DNA is then introduced into a bacterium, where it is replicated many millions of times as the bacterium multiplies
65
genomic library
a set of bacteria, each carrying a different small fragment of the human DNA
contain DNA fragments representing the whole human genome
constructed using restriction enzymes and DNA ligase
reg the figure below, all of the different gray fragments will also be represented in reality
66
complementary DNA (cDNA)
total mRNA is extracted from a selected type of cell, and double-stranded complementary DNA (cDNA) is produced using reverse transcriptase and DNA polymerase
following synthesis of the first cDNA strand by reverse transcriptase, treatment with RNAse leaves a few RNA fragments on the cDNA
the RNA fragment that is base-paired to the 3' end of the first DNA strand acts as the primer for DNA polymerase to synthesize the second cDNA strand
any remaining RNA is degraded during subsequent cloning steps
as different types of cells produce distinct sets of mRNA molecules, each yields a different cDNA library; cells at different stages in their development will also yield different cDNA libraries
67
hybridization
DNA renaturation that is driven by the reformation of the hydrogen bonds between complementary base pairs
hybridization can also occur between complementary strands of DNA and RNA or between two RNAs
can be used to detect any nucleotide sequence of interest by designing a short, single-stranded DNA probe
used to detect complementary RNA molecules in tissue preparations (probes that carry fluorescent or radioactive label); used in cloning of DNA by PCR
68
dideoxynucleoside triphosphate
used in Sanger sequencing
lack 3' –OH; when incorporated into a growing DNA strand, they block further elongation of that strand
the reaction produces are loaded onto a long, thin capillary gel and separated by electrophoresis; a camera reads the color of each band (each color peak represents a nucleotide in the DNA sequence) on the gel and feeds the data to a computer that assembles the sequence
The sequence read from the gel will be complementary to the sequence of the original DNA molecule
69
in situ hybridization
uses single-stranded DNA or RNA probes labeled with either fluorescent dyes or radioactive isotopes to detect complementary nucleic acid sequences within a tissue or an isolated chromosome
used to study the expression patterns of a particular gene or collection of genes in an adult or developing tissue, providing important clues about when and where these genes carry out their functions
70
reporter genes
encodes a protein that can be easily monitored by its fluorescence or enzymatic activity
usually mimics the expression of the gene of interest, producing the reporter protein (green fluorescent protein) when, where, and in the same amount as the normal protein would be made; the expression of protein Y will now be controlled by the regulatory sequences that control the expression of the normal protein X
can also be used to study the regulatory DNA sequences that control the gene's expression; reporters with various combinations of regulatory regions associated with gene X can be constructed
71
CRISPR
system for gene editing in a variety of cells, tissues, and organism
relies on Cas9, a bacterial enzyme that produces a nonsequence-specific double-strand break in a DNA molecules
to direct Cas9 to its target sequence, a guide RNA molecule carried by Cas9 allows the enzyme to search the genome and bind to a segment of DNA with the complementary sequence
CRISPR can generate transgenic organisms and turn selected genes on or off
72
How are transgenic plants made?
1. a disc is cut out of a leaf and incubated in a culture of Agrobacterium with recombinant plasmid
2. the wounded plant cells at the edge of the disc release substances that attract the bacteria, which inject their DNA into the plant cells
3. only those plant cells that take up the appropriate DNA survive and form a callus (mass of relatively undifferentiated cells)
4. the growth factors supplied to the callus induce it to form shoots, which subsequently root and grow into adult plants
73
How can rare proteins be made in large amounts?
a plasmid vector has been engineered to contain a highly active promoter, which causes unusually large amounts of mRNA to be produced from the inserted protein-coding gene
the plasmid is introduced to a cell where the inserted gene is efficiently transcribed and translated
74
What are the functions of the plasma membrane?
cell communication: receptor proteins in the plasma membrane enable the cell to receive signals from the environment
import and export of molecules: channels and transporters in the membrane enable the import and export of small molecules
cell growth and motility: the flexibility of the membrane and its capacity for expansion allows the cell to grow, change shape, and move
75
What is the most common phospholipid in cell membranes?
phosphatidylcholine
built from five parts: hydrophilic head, which consists of choline linked to a phosphate group; two hydrocarbon chains, which form the hydrophobic tails; and a molecule of glycerol, which links the head to the tails; each of the hydrophobic tails is a fatty acid – a hydrocarbon chain with a carboxyl (–COOH) group at one end (glycerol attaches via this carboxyl group
76
triacylglycerols vs phospholipids
fat molecules (triacylglycerols) are entirely hydrophobic, unlike phospholipids (amphipathic)
glycerol is covalently linked to 3 hydrocarbon chains instead of two
77
Why are bilayers self-sealing?
Because free edges exposed to water are energetically unfavorable (hydrophobic tails exposed to water)
a sheet of bilayer folds in on itself and forms a closed vesicle to eliminate the free edges
liposomes are pure phospholipids closed and sealed
78
Where are the new phospholipids made?
the enzymes bound to the cytosolic surface of the ER
the newly added phospholipids are redistributed by scramblase that transfer random phospholipids from one half of the lipid bilayer to the other
this allows the membrane to grow evenly in size and lipid composition
some of the newly assembled membrane will remain in the ER; the rest will be used to supply fresh membrane to other compartments in the cell
79
True or False. Two halves of the bilayer are symmetrical
How is this (not) maintained?
False. most cell membranes are asymmetrical; the two halves of the bilayer have different sets of phospholipids; certain phospholipids are confined to one side of the membrane
flippases in the Golgi membrane remove specific phospholipids from the noncytosolic side and flip them into the cytosolic monolayer
phosphatidylcholine and sphingomyelin are concentrated in the noncytosolic monolayer
phosphatidylserine and phosphatidylethanolamine are on the cytosolic side
glycolipids are exclusively in the noncytosolic monolayer
cholesterole is distributed almost equally in both monolayers
80
What are the various functions of plasma membrane proteins?
transport molecules and ions
* transporter: Na+ pump
* ion channels: K+ leak channel
anchor
* integrins: link intracellular actin filaments to extracellular matrix proteins
detect signals
catalyze reactions
81
What are the different ways membrane proteins associate with the lipid bilayer?
* transmembrane: extend across the bilayer as a single a-helix, as multiple a-helices or as a beta-barrel; integral membrane protein
* monolayer-associated: anchored to the cytosolic half of the lipid bilayer ban an amphipathic a-helix; integral membrane protein
* lipid-linked: on either side of the bilayer by a covalently attached lipid molecule; integral membrane protein
* protein-attached: weak, noncovalent interactions with other membrane proteins (peripheral membrane proteins)
82
How are transmembrane hydrophilic pores formed?
By multiple amphipathic a-helices
the hydrophobic amino acid side chains on one side of each helix come in contact with the hydrophobic lipid tails of the lipid bilayer
the hydrophilic side chains on the opposite side of the helices form a pore
In bacteria,
3 porin proteins (made of beta sheets) associate to form a trimer and form water-filled channels in the outer membrane
83
How can researchers study a membrane protein?
detergents (SDS and Triton X-100) solubilize the membrane and destroy the lipid bilayer by disrupting the hydrophobic associations
this allows separation of the protein of interest from other cell proteins by forming a protein-detergent complex
detergents are amphipathic, lipid-like molecules with a single hydrophobic tail; they aggregate into micelles rather than forming a bilayer
84
What stabilizes the plasma membrane of animal cells?
Cell cortex that is a meshwork of filamentous proteins (made by spectrin dimers and actin molecules) attached to the underside of the membrane
attachment proteins bind to two kinds of transmembrane proteins to attache the network to the plasma membrane
it gives cells their characteristic shape
85
How can a cell restrict the movement of its membrane proteins?
tethered to the cell cortex
tethered to extracellular matrix molecules
tethered to proteins on the surface of another protein
tight junctions (diffusion barriers) restrict proteins to a particular domain
86
glycocalyx
the sugar coating that covers the eukaryotic cells
the carbohydrate-rich layer is made of oligosaccharides attached to membrane glycolipids and glycoproteins and polysaccharide chains on membrane proteoglycans
protects cell surface from mechanical damage, allows WBCs to squeeze through narrow spaces, and prevents blood cells from sticking to one another or to the walls of blood vessels
they have an important role in cell-cell recognition and adhesion; transmembrane protein lectins are specialized to bind to specific oligosaccharide side chains (extremely diverse due to branching, like fingerprinting)
87
Lectins and their role in our immune system
carbohydrates on the surface of neutrophils (WBCs) are recognized by lectins lining the blood vessels at the site of infection
lectins are made by the endothelial cells lining the blood vessel in response to chemical signals emanating from the site of infection
this recognition causes the neutrophils to adhere to the blood vessel wall and migrate from the bloodstream to the infected tissue, where they help destroy the invaders
88
Transporters vs channels
allow inorganic ions and small polar organic molecules to cross the cell membrane
channels:
* form pores across the bilayer
* can exist in either open or closed conformation
* transport only in the open conformation
* controlled by an external stimulus or by conditions within the cell
transporter
* undergo a series of conformational changes
* very selective
* transfer at a much slower rate than channels
* only transporters (pumps) can perform active transports
89
What are the different tactics cells can use to avoid osmotic swelling?
amoeba: periodically eject water that moves into the cell and accumulates in contractile vacuoles
plant cell: cell wall prevents swelling
animal cell: pump out ions
90
what are the three different ways pumps can carry out active transport?
light-driven pumps are mainly found in bacterial cells; they use energy derived from sunlight to drive uphill transport
91
Na+-K+ pump
uses ATP to pump 3 Na+ out and 2 K+ in to keep the cytosolic concentrations of Na+ low and K+ high; this ion gradient can be used to drive active processes in a cell
undergoes a series of conformational changes in an orderly fashion: Na+-dependent phosphorylation and K+-dependent dephosphorylation
92
Ca2+ pump
when a muscle is stimulated, Ca2+ floods into the cytosol from the sarcoplasmic reticulum
the influx of Ca2+ stimulates the cell to contract; to recover from the contraction, Ca2+ must be pumped back into the sarcoplasmic reticulum by the Ca2+ pump
the Ca2+ pump uses ATP to phosphorylate itself, inducing a series of conformational changes
93
glucose-Na+ symport
the pump oscillates randomly between "inward-open" and "outward-open" states. Na+ and glucose can bind to the pump in either of these states, but the pump can transition between them only through an "occluded" state in which both are bound or neither are bound
because the [Na+] is high in the extracellular space, the Na+ binding site is readily occupied in the outward-open state, and the transporter waits for rare glucose to bind
when the transporter randomly flips to the inward-open state, Na+ dissociates (following the concentration gradient). The transporter waits for glucose to eventually dissociate
94
Which transmembrane pumps do animal and plant cells use to drive the active transport of solutes?
in animal cells: Na+ pump
plant, bacteria, and fungi cells: H+ pumped out (in the external environment or into lysosomes/vacuoles)
in animal cells, H+ is pumped out using a Na+-H+ exchanger
95
How do ion channels show selectivity?
by diameter and shape of the ion channel and the distribution of the charged amino acids that line it
an ion channel is narrow enough in places to force ions into contact with the channel wall so that only those ions of appropriate size an charge are able to pass
96
K+ leak channels
generate the resting membrane potential; along with Na+-K+ pump
K+ leaves the cell, moving down its concentration gradient, resulting in a charge imbalance that eventually drives k+ back into the cell
at equilibrium, the effect of the K+ concentration gradient is balanced by the effect of the membrane potential (no net movement of K+ across the membrane)
97
What is the technique used to monitor ion channel activity?
patch-clamp recording
the membrane potential across the isolated patch of membrane is held constant during the recording
In this example, the neurotransmitter acetylcholine binds to the channel protein from a muscle cell, opening the channel and allowing passage of positive ions
but even when acetylcholine is bound to the channel, the channel does not remain open all the time. Instead, it flickers between open and closed states
when acetylcholine is not present, the channel opens very rarely.
98
What are the different types of gated ion channels?
voltage-gated: positively charged amino acids in the channel's voltage sensor domains become attracted to negative charges on the extracellular surface of the depolarized plasma membrane, pulling the channel into its open conformation
ligand-gated (extracellular ligand)
ligand-gated (intracellular ligand)
mechanically-gated: auditory hair cell
99
How is an action potential triggered?
depolarization of a neuron's plasma membrane from -60 mV to -40 mV (the threshold potential); this opens the voltage-gated Na+ channels, further depolarizing the membrane until +40mV, triggering an action potential
100
What are the different conformations of a voltage-gated Na+ channel?
when the membrane is at rest, positively charged amino acids in the voltage sensors of the channel are oriented by the membrane potential in a way that keeps the channel closed
when the membrane is depolarized, the voltage sensors shift, changing the channel's conformation so the channel has a high probability of opening
but in the depolarized membrane, the inactivated conformation is even more stable than the open conformation, so after a brief period spent in the open conformation, the channel becomes temporarily inactivated and cannot open until it goes back to the closed conformation
101
How is an electrical signal converted into a chemical signal at a nerve terminal?
When an action potential reaches a nerve terminal, it opens voltage-gated Ca2+ channels in the plasma membrane, allowing Ca2+ to flow into the terminal
the increased Ca2+ in the nerve terminal stimulates the synaptic vesicles to fuse with the plasma membrane, releasing their neurotransmitter into the synaptic cleft
102
How is a chemical signal converted into an electrical signal?
the released neurotransmitter binds to and opens the postsynaptic transmitter-gated ion channels at the synapse. The resulting ion flows alter the membrane potential of the postsynaptic cell, converting the chemical signal back into an electrical one
103
acetylcholine receptor
of vertebrate skeletal muscle cells open when it binds to acetylcholine
the transmitter-gated ion channel is composed of 5 transmembrane protein subunits that combine to form a transmitter-gated aqueous pore
open conformation: when acetylcholine binds, the channel undergoes a conformational change; negatively charged amino acid side chains at either end of the pore ensure that only positively charged ions can pass, depolarizing the membrane
closed conformation: when acetylcholine is not bound, the channel pore is blocked by hydrophobic amino acid side chains in the gate
104
optogenetics
light-gated ion channels control the activity of specific neurons in a living animal
the gene encoding channelrhodopsin is introduced into a subset of neurons in the mouse hypothalamus
when the neurons are exposed to blue light using a tiny fiber-optic cable implanted into the animals' brain, channelrhodopsin opens, depolarizing and stimulating the channel-containing neurons
optogenetics allows investigators to study where and how genes function in model organisms to get a better understanding of the molecular and cellular basis of human behavior
105
What are the inputs and outputs of glycolysis?
How many steps are there and what are the main characteristics of these steps?
glucose + 2 ATPs --\> 2 NADH + 4 ATP + 2 pyruvate
10 steps
step 1-3: energy investment
step 4-5: cleavage of the six-carbon sugar to 2 3-carbon sugars
steps 6-10: energy generation
106
What happens to pyruvate in the absence of oxygen?
Pyruvate is broken down by fermentation into lactate (muscle) or CO2 and ethanol (yeast)
fermentation restores the NAD+ consumed in step 6 of glycolysis
yields much less energy than if the pyruvate were oxidized in mitochondria
2 pyruvates --\> 2 NAD+ + 2 lactate
2 pyruvates --\> 2 CO2 + 2 ethanol + 2 NAD+
107
What happens to pyruvate in the presence of oxygen?
converted into acetyl CoA and CO2 by pyruvate dehydrogenase in the mitochondrial matrix to generate NADH
the citric acid cycle catalyzes the complete oxidation of acetyl CoA
one turn of the cycle produces 3 NADH, 1 GTP, and 1 FADH2 and releases 2 CO2
108
besides glucose, what other organic molecules are converted to acetyl CoA in the mitochondrial matrix?
fatty acids derived from triacylglycerol (fat) are also converted into acetyl CoA
lipases hydrolyze the ester bonds when fatty acids are needed for energy; the released fatty acids are activated and subsequently oxidized
each oxidation cycle shortens the fatty acyl CoA by 2 carbons and generates 1 FADH2, 1 NADH, and 1 acetyl CoA
109
oxidative phosphorylation
completes the catabolism of food
generates the bulk of the ATP made by the cell
activated carriers donate their high-energy electrons to an electron-transport chain in the inner mitochondrial membrane. H+
This electron transfer pumps H+ across the inner membrane. The resulting proton gradient is then used to drive the synthesis of ATP through the process of oxidative phosphorylation
110
gluconeogenesis
makes glucose from pyruvate
there are three steps in glycolysis that are so energetically favorable that they are irreversible (steps 1, 3, and 10); to bypass those steps, gluconeogenesis uses a special set of enzymes
In step 3 of glycolysis, phosphofructokinase uses ATP to phosphorylate fructose 6-phosphate; in gluconeogenesis, fructose 1,6-bisphosphatase removes phosphate from this intermediate to generate fructose 6- phosphate
coordinated feedback regulation of these two enzymes helps a cell control the flow of metabolites toward glucose synthesis or glucose breakdown
phosphofructokinase is activated by by-products of ATP hydrolysis and is inhibited by ATP; fructose 1,6-bisphosphatase is regulated in the opposite direction
111
glycogen
a branched polymer of glucose (storage) in glycogen granules in the cytoplasm of liver and muscle cells; granules also have enzymes required for glycogen synthesis (synthetase) and breakdown (phosphorylase)
112
where are fats stored?
fats stored in the form of lipid droplets in the cytoplasm of adipocytes
far more important storage material than glycogen; glycogen is bigger in mass and releases half the energy as the oxidation of a gram of fat
any excess glucose is used to make glycogen or fat; fatty acids cannot be converted to sugars
113
where do plant cells store their starch and fats?
in chloroplasts
