29 - Translation II

Question 1

What are the three binding sites in prokaryotic ribosomes?

Answer 1

• E (exit)
• P (peptidyl)
• A (aminoacyl)

Question 2

What are the steps of translation synthesis in prokaryotes? (detailed with initiation factors) (4)

Answer 2

1. IF3 and IF1 bind to the 30S subunit
2. The fmet-tRNA accompanied by IF2 base pairs with the start codon
3. The 50S subunit associates with the 30S subunit
4. IF2 hydrolyzes GTP and IF1, IF2 and IF3 dissociate to leave the initiation complex

Question 3

What are the three initiation factors needed in prokaryotic translation initiation and what do they do?

Answer 3

IF1 - Prevents premature binding of tRNA to A site

IF2 - Facilitates binding of fMet-tRNA to 30S subunit

IF3 - Enhances specificity of P site for fMEt-tRNA

Question 4

What are the additional initiation factors that eukaryotes require (in addition to prokaryotic initiation factors)? (5)

Answer 4

eIF1a
eIF4e, eIF4g, eIF4f
eIF5, eIF5b

Question 5

Prokaryotic translation requires three elongation factors for elongation? What are they? What else is needed?

Answer 5

EF-Tu
EF-Ts
EF-G

GTP needed

Question 6

What are the four steps in prokaryotic elongation (details about elongation factors)?

Answer 6

• Incoming aminoacyl-tRNA binds GTP-bound EF-Tu
• This complex binds to A site of 70S initiation complex
• GTP is hydrolyzed and EF-Tu-GDP is released
• EF-Ts participates in recharging EF-Tu-GDP with GTP

Question 7

Describe the peptide bond synthesis step of prokaryotic translation

Answer 7

• Nucleophilic attack by the N of the alpha-amino group (NH2) of the aminoacyl-tRNA in the A siteon the electron deficient C of the carbonyl group of the ester linkage of peptidyl-tRNA in the P site
• 23S rRNA catalyzes the reaction which involves ribose hydroxyl groups on the tRNA substrate

Question 8

What part of the tRNA catalyzes formation of the peptidyl bonds?

Answer 8

Ribose hydroxyl groups on the 23S tRNA substrate, specifically the 2’-OH of the peptidly-tRNA bound in the P site of the ribosome

Question 9

Describe the translocation step of prokaryotic translation elongation

Answer 9

• GTP bound EF-G (the translocase) binds the A site
• Hydrolysis of the GTP leads to a conformational shift ni the ribosome that moves the mRNA and the tRNAs by 1 codon
• Following translocation and tRNA release, the ribosome is ready to accept the next aminoacyl tRNA (aa-tRNA) and repeat the cycle
• The cycles will continue until a termination codon is reached

Question 10

What are the three protein release factors that recognize stop codons for translation in bacteria?

Answer 10

RF1: UAA and UAG
RF2: UAA and UGA
RF3: GT{ase that promotes release

Question 11

What are the steps of termination of translation in bacteria?

Answer 11

• Release factor binds to stop codon
• Polypeptidal-tRNA link hydrolyzed
• Release factors dissociate and are replaced by GTP-EF0G and ribosome recycling factor (RRF)
• Hydrolysis of GTP dissociates 50S subunit
• IF-3 replaces GDP-EG-G and RRF leaving 30S-IF3 complex ready for next round of protein synthesis

Question 12

How do EF-G (elongation factor) and RF (release factor) fit into a site on the ribosome fitted for a tRNA?

Answer 12

Release factor RF2 is structurally similar to a domain in elongation factor EF-G, that in turn in structurally similar to the tRNA portion of a ternary complex of aa-tRNA/EF-Tu/GTP

Molecular mimicry: EF-G and RF ‘mimic’ a trnA in binding to the A site of the ribosome in response to a translation stop codon. In spire of their very different nature (proteins vs. tRNA)

Question 13

How does translation differ in eukaryotes and prokaryotes differ? (broadly..)

Answer 13

• Initiation of translation is more complex in eukaryotes and requires more protein factors
• Mechanisms of control of translation are also more developed in eukaryotes

Question 14

Is gene expression at the translational level specific or general?

Answer 14

Translational regulation can be general (affecting all genes) or specific (one or a group of genes)

Question 15

What is the stringent response in E. coli?

Answer 15

An example of gene expression regulation at the translational level

If the cell begins to starve from a lack of amino acids, tRNAs are empty. There is induction of ppGpp synthesis which reorganizes the cell to endure tough conditions.

Question 16

Defective mRNAs can cause a variety of problems, such as producing faulty proteins (some can be toxic) or stall the ribosome. What is one way to cope with this in bacteria?

Answer 16

Bacteria have a system to solve stalling ribosomes when they reach the end of a truncated mRNA.

• tmRNA (combination of tRNA and mRNA) displaces the original faulty mRNA in ribosomes at the A site, elongation continues with emerging protein tagged for degradation by proteases

Question 17

How do eukaryotes regulate translation when there are non-stop mRNAs?

Answer 17

• The non stop mRNA is causing excessive poly(Lys) to be translated by poly(A) tail
• ski7 recognizes this and binds to the ribosome
• ski7 recruits an exosome which degrades the mRNA, it also promotes disassembly of ribosome and assembly of exosome and protease
• The lysine repeats on the polypeptide will tag it for degradation by a protease

Question 18

How do eukaryotes regulate translation when they have premature stop codons in their mRNA?

Answer 18

Nonsense mediated mRNA decay

• Upf1 and Upf2 bind to the mRNA downstream of the premature stop codon (recognize stalled sequence)
• A decapping enzyme comes along and decaps the mRNA
• An endonuclease degrades the mRNA from the 5’ end to 3’ end

Question 19

When are mRNAs with premature translation termination codons (PTCs) targeted for degradation?

Answer 19

After the first round of translation

Question 20

How are premature translation termination codons recognized?

Answer 20

Stop codons need to be within a certain distance from the poly(A) binding protein to be recognized as authentic.