IT3: Translation

Question 1

What is the Shine Dalgarno sequence?

Answer 1

The ribosomal binding site in bacteria.

Question 2

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

Answer 2

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 3

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

Answer 3

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 4

How is translation initiation regulated in bacteria?

Answer 4

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 5

How is translation initiation regulated in eukaryotes?

Answer 5

• 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 6

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

Answer 6

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 7

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

Answer 7

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

Question 8

How do tRNAs recognize the correct amino acid?

Answer 8

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 9

What is the double-sieve mechanism of tRNAs?

Answer 9

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 10

What is a wobble interaction?

Answer 10

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 11

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

Answer 11

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 12

What are A-minor interactions?

Answer 12

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 13

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

Answer 13

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 14

Define proofreading

Answer 14

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 15

Define accommodation

Answer 15

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

Question 16

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

Answer 16

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 17

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

Answer 17

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: conformational changes in the ribosome locks only cognate tRNA into the tRNA (proofreading).

Question 18

How are peptide bonds formed during translation?

Answer 18

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 19

What is the ternary complex?

Answer 19

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 20

How is the peptide chain elongated?

Answer 20

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 21

How is translation initiated in bacteria?

Answer 21

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 22

How is translation terminated in bacteria?

Answer 22

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.