IT3 - Gene Expression

Question 1

How does the trigger loop contribute to accuracy?

Answer 1

It eases the barriers to backtracking - a mechanism for removing mis-incorporated nucleotides.

Question 2

How is translation initiation regulated in eukaryotes?

Answer 2

• Controlling the interaction between the ribosome, the ternary complex, and the 5’ CAP.
• Interfering with scanning using RNA secondary structure supported by RNA binding proteins.
• As the mRNA template is a loop, with links between the poly(A) binding protein and CAP binding proteins, sequences in the 3’ UTR also control translation initiation.
• Many kinases target factors required for translation.

Question 3

Define accommodation

Answer 3

The movement of the amino-acylated end of the tRNA into the P-site for peptide bond formation.

Question 4

What are cognate vs near-cognate vs non-cognate interactions?

Answer 4

A cognate interaction is when the anticodon of the tRNA is precisely matched to the codon on the mRNA.
Near-cognate interactions occur when the anticodon isn’t a perfect match, due to wobble base pairing. This may result in the addition of incorrect amino acids.
Non-cognate interactions occur when the anticodon doesn’t match the codon at all, often due to misacylation of tRNA or mutations.

Question 5

How are cognate and wobble interactions distinguished at the A-site?

Answer 5

A1493 forms A-minor interactions at position 1 that are different with a wobble interaction.
There are restrictions on the allowed geometries of the first two nucleotides of the codon.

Question 6

What are the conformations of the RNAP clamp?

Answer 6

It is initially in a more open position, but after DNA has been melted, it gets repositioned to keep the DNA in the active site tightly (i.e., it ‘clamps’ down onto the DNA).

Question 7

What is a super enhancer/LCR?

Answer 7

A large cluster of enhancers that control the expression of genes that are critical for cell identity and differentiation. The best characterized of these are the beta-globin loci.

Question 8

What are the shared features of amino-acyl tRNAs (except initiation tRNA)?

Answer 8

1. Secondary structure, including modified nucleotides which are changed after transcription.
2. Tertiary structure

Question 9

What 3 models explain how a distal enhancer could regulate a promoter?

Answer 9

1. Stable contact model
2. Kiss and run mode: co-activators deposit PTMs at the promoter and TFs are transferred.
3. Communication by diffusion model: TFs activated at the enhancer then diffuse to the promoter (likely involves condensates).

Question 10

What’s the difference between EFTu-dependent and -independent proofreading?

Answer 10

Dependent: EFTu-GTP is hydrolysed if the correct tRNA is used, releasing the tRNA for peptide bond formation. Hydrolysis is inhibited if the tRNA is incorrect (initial selection).
Independent: After GTP hydrolysis, the ribosome undergoes conformational changes that selectively stabilise only the cognate tRNA in the A site, effectively ‘locking’ it in place for peptide bond formation. Non-cognate or near-cognate tRNAs are rejected during this step.

Question 11

Describe Rho-dependent termination.

Answer 11

Rho is an ATPase that travels with some elongating RNAPs and pulls the RNA out of RNAP, terminating transcription by extraction.

Question 12

What is the pre-initiation complex?

Answer 12

The preinitiation complex is a complex of approximately 100 proteins that is necessary for the transcription of protein-coding genes in eukaryotes and archaea. The preinitiation complex positions RNA polymerase II at gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site for transcription.

The minimal PIC includes RNA polymerase II and six general transcription factors. Additional regulatory complexes (such as the mediator coactivator and chromatin remodeling complexes) may also be components of the PIC.

Question 13

What is the double-sieve mechanism of tRNAs?

Answer 13

A model that explains the rarity of misacylation of amino acids by proposing that an amino acid larger than the correct one is rarely activated because (1) it is too large to fit into the active site of the tRNA synthetase (first sieving), and (2) the hydrolytic site of the same synthetase is too small for the correct amino acid (second sieving). Thus, an amino acid smaller than the correct one can be removed by hydrolysis.

Question 14

What is the ternary complex?

Answer 14

EFTu-aatRNA-GTP. This is delivered to the ribosome where the aatRNA is matched to the appropriate mRNA codon and incorporated into the growing protein chain.

Question 15

How can cysteine levels alter transcription of the ubiGmccBA operon?

Answer 15

The 5’UTR is able to form either a terminator or anti-terminator structure depending on the cys-tRNA presence. The presence of cys-tRNA signals that there are high levels of cysteine, and so the terminating structure is formed.
If the cell is low in cys-tRNA, but high in SAM, the uncharged tRNA prevents terminator loop formation, and the structure is altered to allow termination of the antisense strand, giving high expression of structural genes to convert SAM into cysteine.
If there’s low cys-tRNA levels and low SAM levels, the uncharged tRNA binds to the 5’UTR and prevents terminator structure formation. This allows for synthesis of the operon that encodes enzymes needed to synthesize cysteine from methionine.

Question 16

How is translation initiated in bacteria?

Answer 16

The Shine-Dalgarno sequence on the mRNA positions the 30S subunit so that the AUG is in the P site. Initiator tRNA (f-Met) binds in the P site with the help of GTPase, IF2. IF1 blocks the A site and IF3 blocks the E site.
Once the ribosome is fully assembled, the GTP-bound initiation factor IF2 hydrolyzes its GTP to GDP and Pi, triggering the release of the IFs and the start of protein synthesis.

Question 17

How may stochastic gene transcription arise in eukaryotic cells?

Answer 17

A major source of variation in eukaryotes is chromatin which can control access of TFs to their binding sites.

Question 18

What is the role of the sigma70 3.2 loop?

Answer 18

Protrudes into the RNAP active site, stabilizing the binding of the initiating nucleotide substrate and promoting abortive initiation by blocking the path of the nascent RNA into the exit channel.

Question 19

What is a proto-enhancer?

Answer 19

Modular components of an enhancer that each bind a TF. Each proto-enhancer is made up of an enhanson - the fundamental units of enhancer function. e.g., the SV40 enhancer.

Question 20

Why do bacteria need to regulate gene expression at the level of translation?

Answer 20

Many genes in bacteria are encoded into operons, under the control of one promoter. Each gene is equally represented on the mRNA, so the cell uses translational control to produce different levels of protein from the same operon.

Question 21

What are co-activators and co-repressors? What 2 models describe how they might work?

Answer 21

Co-activators such as Mediator facilitate interactions between TFs bound to proximal or distal enhancers and the PIC bound to the core promoter. Co-repressors prevent these interactions.
1. The sequential model of activation: a cell-specific and a general co-activator are proposed to form direct interactions captured by a cohesin loop.
2. Condensates: local high concentrations of phase-separated molecules provide a regulatory mechanism to compartmentalize biochemical reactions, such as transcription.

Question 22

Describe Rho-independent termination.

Answer 22

(Intrinsic termination) occurs in bacteria using a terminator motif that’s present in the DNA template. This motif usually contains an inverted repeat followed by a U-rich tract, resulting in the formation of a stem-lop in the RNA transcript. This causes pausing of RNAP and destabilization of the RNAP:DNA:RNA complex.
Destabilization triggers release of mRNA, leading to termination.

RNA that’s tightly associated with ribosomes can’t form the pausing stem-loop structures for termination.

Question 23

How is translation initiation regulated in bacteria?

Answer 23

Regulation of translation in bacteria at initiation occurs by preventing the ribosome from recognizing the Shine Dalgarno site and AUG by RNA structure stabilized proteins, metabolites, or tRNAs.

Question 24

Why may RNAP pause and what can this lead to?

Answer 24

1. Rewinding of the DNA supplies little energy (A-T rich) and unwinding of the downstream DNA requires more energy (G-C rich), reducing rate of elongation and pausing.
2. Weak base-pairing in the hybrid region compared to upstream e.g., A-U.
   In these circumstances, the DNA:RNA hybrid may return upstream whilst the downstream edge rewinds, leading to backtracking.
   This causes displacement of the 3’ end from the active site and so this end must be cleaved to generate a new 3’OH for synthesis to continue.

Question 25

Define proofreading

Answer 25

The process by which, after initial recognition, cognate vs near-cognate aatRNAs are discriminated in the A-site. EFTu's GTPase is activated, and EFTu-GDP is released. This allows the ribosome to undergo structural changes in preparation for accommodation.

Question 26

What are the roles of the 3 types of eukaryotic RNAPs?

Answer 26

Pol I transcribes rRNA genes. Pol II transcribes mRNA. Pol III transcribes tRNAs and snRNAs.

Question 27

Why do eukaryotes regulate gene expression at the level of translation?

Answer 27

Allows fine tuning of the levels of proteins and fast responses to regulatory signals. During early development there's no transcription, and so events such as the cell cycle rely on translational control of the production of proteins.

Question 28

How does RNAP find and bind to a promoter in prokaryotes?

Answer 28

1. RNAP binds on and off to random DNA sequences, scanning until it finds promoter sequences. 2. -10 and -35 need to be contacted at the same time, resulting in distortion of the DNA. 3. Introducing this torsional stress forces the DNA to melt to relieve the strain, temporarily becoming single-stranded. This is the open complex.

Question 29

State the 3 methods used in bacteria to transduce a signal.

Answer 29

Anti-sigma factors 2-component regulatory systems Individual activators and repressors

Question 30

How does a promoter differ from an enhancer?

Answer 30

Promoter: - Binding site for RNAP and other TFs. - Located near the TSS - H3K4me3 and H3K27ac Enhancer: - Bind TFs to enhance transcription - Located upstream or downstream of TSS - Can confer tissue- or developmental-specific expression patterns. - H3K4me1 and H3K27ac

Question 31

What is the elemental pause state of RNAP?

Answer 31

A step during elongation where the enzyme undergoes brief stalling. This is a highly conserved process that allows for the recruitment of various elongation factors and regulatory proteins to modulate RNAP activity e.g., NusA and Spt5. Pausing arises when nucleic acid interactions in the elongation complex lead to a loosening of the clamp domain and incomplete translocation, trapping RNAP in the paused state. The hairpin-stabilized pause is generated from an elemental pause when the nascent RNA folds into a hairpin that jams open the clamp domain and traps the trigger loop in an inactive conformation.

Question 32

How are peptide bonds formed during translation?

Answer 32

The peptidyl transferase activity of the ribosome catalyzes the formation of a peptide bond by transferring the amino group of the amino acid in the A site to the carbonyl group of the amino acid in the P site. This reaction results in the release of the tRNA from the P site and the transfer of the peptide chain to the tRNA in the A site. The ribosome then moves along the mRNA to the next codon, and the process is repeated. A key 2'OH on A76 makes an essential contribution to this reaction, likely through a proton wire mechanism.

Question 33

What is a wobble interaction?

Answer 33

The wobble hypothesis proposes that the 3rd nucleotide can sometimes form non-standard base pairs. This means that a tRNA with a given anticodon can recognize more than one codon, as long as the codons differ only in the third position.

Question 34

How does sigma70 melt the DNA at the lac operon?

Answer 34

The lac operon has a non-consensus sequence - instead of the Pribnow box being AT rich, it contains a GC link that prevents sigma70 from binding. Activators Crp/CAP improve the Kb by providing additional contacts for RNAP, and k+2 by further distorting/bending the DNA.

Question 35

Describe abortive initiation

Answer 35

Abortive initiation is an obligatory step in both bacteria and eukaryotes that helps RNAP to ensure it has properly positioned itself at the TSS. RNAP synthesizes ~9 nucleotides in its holoenzyme form, but due to the sigma70 3.2 loop blocking the exit channel, the DNA collects within a pocket of the enzyme. This causes distortion and increases the strain of the interaction between the DNA and the enzyme. Relaxation can be achieved by releasing RNA, thus aborting transcription. If everything is correct, the sigma factor is released and RNAP enters elongation.

Question 36

How is the peptide chain elongated?

Answer 36

GTP-bound EF-G induces a conformational change that causes the ribosome to move along mRNA. Hydrolysis of GTP releases EF-G for the next cycle of elongation.

Question 37

Describe the mechanism of elongation in transcription.

Answer 37

Spt5 (NusG in bacteria) is an elongation factor that binds the polymerase clamp to help stabilize the interaction between RNAP and the DNA, enhancing processivity and preventing premature termination. It can only associate upon promoter escape because its binding site is occupied in the initiation complex. During the NAC, an NTP substrate binds to the open active center before occupying the insertion site as the trigger loop folds to close the active center. This leads to catalytic nucleotide incorporation. Release of PPi may cause trigger loop unfolding and opening of the active site. The bridge helix works with the trigger loop to shift the newly integrated NTP out of the active site.

Question 38

How do tRNAs recognize the correct amino acid?

Answer 38

Some aa-tRNAs are able to undergo hydrolytic editing. The recognition system requires two steps of discrimination independently, due to the minor differences in energy between many amino acids. These are separated by an irreversible step - the hydrolysis of ATP. 1. Pre-transfer editing: prevents mischarged aa-tRNA formation through hydrolysis of misactivated aminoacyl-adenylates before transfer. 2. Post-transfer editing: directly targets the malformed aa-tRNA for deacylation

Question 39

What is the Shine Dalgarno sequence?

Answer 39

The ribosomal binding site in bacteria.

Question 40

How is translation terminated in bacteria?

Answer 40

1. As the ribosome moves along the mRNA, it eventually encounters a stop codon in the A site of the ribosome. 2. RF1/RF2 bind the A site and the peptide is released. 3. RF3-GTP binds and hydrolyzes GTP to release RF1/RF2. 4. Ribosome release factor binds with EF-G, releasing RF3. 5. GTP hydrolysis occurs to help the ribosome dissociate.

Question 41

What is the molecular basis of discrimination of cognate and near-cognate tRNAs at the A-site during translation?

Answer 41

Decoding. 1. G530 stabilizes the cognate codon-anticodon helix, initiating latching of the decoding center. 2. This results in the closure of the 30S subunit and docking of EFTu with the Sarcin-Ricin loop. 3. SRL activates EFTu for GTP hydrolysis, enabling accommodation of the tRNA. Near-cognate complexes fail to induce the G530 latch, unless they tautomerize.

Question 42

How does attenuation differ from antitermination?

Answer 42

Attenuation: - Terminates transcription - Occurs when transcription is slower than translation, allowing the ribosome to catch up to RNAP, causing formation of an RNA secondary structure that prematurely terminates transcription - Can be mediated by ribosomes, proteins, or uncharged tRNA In trp operon, NusG and NusA aid termination by stabilizing the hairpin and stopping translocation. Antitermination: - Allows transcription to continue beyond a termination site - Can occur in response to environmental or cellular signals, mediated by factors such as NusA and Spt5 - Factors prevent formation of termination structure

Question 43

What has looking at mutation rates told us about tRNA synthetase?

Answer 43

Mutations that lead to mis-charging can be in the anticodon, the D-loop, or in the acceptor stem of the tRNA suggesting that the synthetase inspects the whole conformation of the tRNA as well as details of base recognition. The presence of modified nucleotides also suggests that local details of shape are important for recognition.

Question 44

Compare sigma70 and sigma54

Answer 44

Sigma70 recognizes promoters at -10 (Pribnow box) and -35. It can initiate transcription without assistance (except at the lac operon). Sigma54 is involved in nitrogen-regulated genes, binding at -12 and -24 and requires bacterial enhancer binding proteins to 'melt' the DNA, using ATP. In this way, bEPB acts like eukaryotic TFIIH as both require ATP.

Question 45

How does bacterial RNAP differ from eukaryotic RNAP?

Answer 45

Bacteria: - Consists of a core enzyme and a sigma factor that makes up the holoenzyme - Can initiate transcription on its own - Primarily regulated by sigma factor availability Eukaryotic: - Consists of several subunits that form the core enzyme, along with accessory factors - Requires additional factors (e.g., GTFs and Mediator) to initiation transcription - Regulated by TFs, chromatin modifiers, and signaling pathways

Question 46

What are A-minor interactions?

Answer 46

A-minor interactions are noncanonical hydrogen bonds that play a significant role in RNA structure and function to help anchor the correct codon-anticodon interaction in at the A site. These interactions involve G530, A1492 and A1493.

Question 47

Describe the steps of how sigma70 leads to elongation.

Answer 47

1. Sigma70 is required to melt the DNA 2. Loop 3.2 of sigma70 is required for the formation of the first phosphodiester bond 3. It promotes abortive initiation but prevents the move to the elongating form of RNAP. 4. Must be displaced from RNAP to elongate.