Translation 2 - Mechanism Flashcards
Where do IF-1 and IF-3 bind in prokaryotic initiation?
The small 30S subunit. IF-1 binds to the A site.
What does IF-3 binding do?
Prevents premature assembly of the 30S subunit with the 50S subunit.
How does mRNA bind to the 30S subunit?
By using the Shine Dalgarno sequence.
Where is the Shine Dalgarno sequence found?
8 bases upstream of AUG.
How is the first Met-charged tRNA brought into the ribsome?
Brought into the P site on the small subunit with IF2-GTP.
What does GTP hydrolysis drive during initiation?
A major conformational change that allows the large subunit and the small subunit to join. All initiation factors are then released.
What are the differences between eukaryotic and prokaryotic translation initiation?
- Eukaryotes require more initiation factors. - Eukaryotes don’t have a Shine Dalgarno sequence.
How is the ribosome positioned in eukaryotic translation?
By interactions with both ends of the mRNA- the 5’cap and the poly(A) tail.
How is the second charged tRNA added?
It is brought into the A site by EF-TuGTP. GTP hydrolysis causes a conformational change, locking the tRNA in place and releases EF-TuGDP.
How is EF-TuGTP regenerated during translation elongation?
Regenerated by EF-Ts, a guanine exchange factor.
How is peptide bond formation catalysed during elongation?
Catalysed by 23S rRNA. Occurs via nucleophilic attack of the C=O group of the peptide in the P site, by the NH2 group of the amino acid in the A site.
How is the ribosome moved along to the next codon after peptide bond formation?
Requires EF-G and GTP hydrolysis. Moves the uncharged tRNA into the E site and the peptidyl-tRNA into the P site, leaving the A site available for the next charged tRNA.
Compare translation elongation in prokaryotes and eukaryotes.
Occurs via the same mechanism. Eukaryotes have analogous elongation factors; eEF1α, eEF1βγ and eEF2.
How is translation terminated?
A release factor binds to the A site and the ester bond between the C=O of the amino acid and the OH of the tRNA is hydrolysed. All of the ribosome subunits, uncharged tRNA and release factors dissociate.
Compare translation termination in prokaryotes and eukaryotes.
In prokaryotes there are different release factors to recognise different stop codons- RF1 for UAA, RF2 for UAA and UGA. In eukaryotes there is only one release factor- eRF.
How can translation be inhibited?
By antibiotics that mimic translation factors, e.g. Tetracyclins block the A site.
What suggests that there are structural differences between the eukaryotic and prokaryotic ribosomes?
There are antibiotics that specifically inhibit only one. For example, chloramphenicol blocks peptidyl-transferase but cycloheximide only blocks the eukaryotic peptidyl-transferase.
How does EF-Tu improve translation accuracy?
As it binds the charged tRNA, it checks that the amino acid and the tRNA are correctly matched. Charged tRNAs are in a bent conformation when bound to EF-TuGTP:- allows codon pairing, but not addition of the amino acid to the peptide- GTP hydrolysis will only occur if the codon and anticodon are correctly matched.
What is the level of translation accuracy in a ribosome?
99.99% accuracy.
What happens to incorrectly matched tRNAs?
They dissociate quicker than correctly matched tRNAs due to weaker binding to the codon and are not used in protein synthesis.
Why can EF-G bind the A site of the ribosome?
The EF-G protein, also called translocase, can bind the A site of the ribosome because its structure resembles the EF-Tu/tRNA complex.
Why is the EF-G (translocase) protein needed for movement of the ribosome along the mRNA?
In binding the A site, EF-G protein displaces the peptidyl-tRNA bound there.
How is mischarging resolved in isoleucyl- tRNA synthetase?
Has an editing site that fits val-tRNA (Ile) which cleaves the valine. This prevents mischarging due to the similarity of isoleucine and valine.
What amino acids do class I aa-tRNA synthetases usually correspond to?
Larger and more hydrophobic amino acids.
Describe the Class I aa-tRNA synthetases.
Can be monomeric or dimeric. Can use either the 3’OH or the 2’OH of the adenine during aminoacylation.
Describe the class II aa-tRNA synthetases.
Can be dimeric or tetrameric. Always use the 3’OH in the adenine for aminoacylation.
What is the key difference between prokaryotic and eukaryotic aa-tRNA synthetases?
Prokaryotic aa-tRNA synthetases are individual proteins whereas in eukaryotes aa-tRNA synthetases act as multienzyme complexes, e.g. GluProRS
What happens in bacteria that lack the aa-tRNA synthetase for glutamine?
Glu-tRNA synthetase charges the tRNA(Gln) with glutamic acid. Glutamic acid is then converted to glutamine by another enzyme.
What is ribosome ratcheting and when does it occur?
Rotation of the 30S subunit by approximately 6 degrees. Occurs after peptide bond formation.
When is ribosome ratcheting reversed?
Upon EF-G(GTP) binding and GTP hydrolysis.
What is the purpose of ribosome ratcheting?
The conformational change in the ribosomal subunits affects the binding affinity of the E/P/A sites, e.g. When reversed, A is in high affinity and E in low affinity, meaning uncharged tRNA can be released from E and new aa-tRNA can bind in A.
How does ribosome ratcheting promote translocation of uncharged tRNA from the A site to the E site (once peptide bond has formed)?
E site is in a high affinity state and binds uncharged tRNA. A site is in a low affinity state.
What is the role of the ribosome recycling factor in bacteria?
Splits the inactive ribosome into its constituent subunits, using EF-G and GTP. This is transient and ribosomal subunits will reassociate unless IF-3 is present.
Are the classes of aa-tRNAs conserved?
Yes, each class contains the same 10aas in most organisms.
What are the analogous elongation factors found in eukaryotes?
eEF-1a analogous to EF-TU
eEF-1by analogous to EF-Ts
eEF-2 analogous to EF-G
