Lecture 3: Peptide bond formation Flashcards
What is the overall mechanism of the ribosome?
1) Aminoacyl tRNA binds as a complex with EF-Tu and GTP.
2) GTP is cleaved and EF-Tu, GDP and Pi is released. Aminoacyl tRNA is left in the A site.
3) Peptide from the P site is transferred from an ester linkage with tRNA to a peptide linkage by the peptidyl transferase.
4) Peptidyl-tRNA is transferred from the A site to the P site by EFG. GTP hydrolysis occurs.
The initial selection and the proofreading are separated by irreversible steps.
How does proofreading occur?
Discrimination occurs in 2 consecutive steps. Ribosomal proteins such as S13 and L27 are actively involved in the proofreading of tRNA. Protein tails from the small and large ribosomal subunits cause strengthening of tRNA binding.
• Proofreading starts with monitoring the entire anticodon loop of tRNA. This is done from 16S rRNAs (helix 18: G530 and helix 44: A1492/1493) of the small subunit and 23S rRNA (helix 69; A1913) of the large subunit with involvement of the magnesium ions.
• The elbow region of tRNA is scanned by rRNA (helices 38 and 89) and ribosomal proteins choose whether to accommodate the acceptor end of the tRNA in the PTC or not.
How does accommodation occur?
- Structure changes from I and I (30S open) to III (30S closed). The 30S shoulder moves towards the 50S subunit by around 4 Angstroms. This shifts the GTPase domain of EF-Tu by 8 Angstroms in order to bind the SRL.
- Accommodation is when the aa-tRNA moves into the peptidyl (transferase centre (PTC) after release from EF-Tu.
- tRNA proceeds in 3 steps. First the aa-tRNA elbow, then the acceptor arm and then the codon end entry into the PTC.
- Accommodation is very efficient, virtually all cognate aa-tRNA enters the PTC.
- Residues in the 23S rRNA protect the P-site peptidyl tRNA from premature hydrolysis until the A-site aa-tRNA is accommodated.
- 23S ribosomal residues sterically protect the P-site bound acyl donor (peptidyl-tRNA) from premature hydrolysis when the A site is occupied.
- Accommodation induces a conformational change that reorganises the active site for transfer of the peptidyl group to the amine nucleophile.
Explain the peptidyl transferase reaction
- Peptide is transferred from its linkage to the tRNA to make a peptide link with the amino group of the aa-tRNA in the A site.
- No additional energy is needed.
- EF-Tu-GDP must have already dissociated. This is a slow step.
- Peptidyl transferase activity is known to be a property of the 50S subunit.
- Catalytic centre is only RNA.
How do antibiotics work at the PTC of the 50S subunit?
- Chloramphenicol: prevents peptide formation by overlapping with the binding site of the A-site tRNA.
- Clindamycin: prevents peptide formation by sterically blocking A-site tRNA positioning at the PTC.
- Sparsomycin: stabilises P-site tRNA binding by promoting translocation of A-site tRNAs into the P-site and inhibiting peptidyl transfer.
How does the peptidyl transferase reaction work on an energetic and catalytic level?
The PT reaction is accelerated by a factor of 4 million. However, this is not done through chemical catalysis.
• Proton transfer during the reaction involves ribose hydroxyl groups on the A76 of the tRNA substrate.
• Mutations show very small chemical contribution. Residues form a hydrogen bond network.
• The mechanism involves an intra-reactant proton shuffling via the P-site peptidyl tRNA 2’ oxygen after the attack of the A-site amino group on the P-site ester. This may work directly or indirectly through one of more active-site waters.
• Nitrogen nucleophiles react with carboxylate esters to form amide bonds. Tetrahedral intermediate forms. Proton is lost from the nucleophilic nitrogen. Intermediate collapses to form the amide.
• The ribosome puts the reactive groups in the right proximity and orientation.
• It may also work by shielding the reaction against water, helping the proton shuttle form the leaving group, reducing the activation energy of the highly polar transition state or a combination of all of these.
How does translocation work?
Translocation is when the peptidyl-tRNA is moved to the P site in order to free up the A site.
• After peptidyl bond creation, deacetylated tRNA is in the P site. A lengthened peptidyl-tRNA is in the A site (proven with puromycin).
• EGF is required to move the peptidyl tRNA into the P site and free up the A site.
• Deacetylated tRNA is moved from the P site into the E site.
• As peptidyl-tRNA moves, mRNA also moves and a new codon appears in the A site.
• EFG requires GTP hydrolysis to create a conformational rearrangement to the ribosome.
• EF-G prevents back reaction and lowers kinetic barriers by binding to the A site.
• Translocation occurs slowly without any protein factors.
• EF-G is a mimic of the ternary complex (they both bind the A site). EF-G disrupts interactions between the decoding centre and codon-anticodon duplex.
• After translocation, the ribosome can bind another EF-Tu-GTP-aatRNA in the A site. It cannot bind another EFG.
How does the exit tunnel work?
The exit tunnel stretches from the PTC and ends at the back side of the large subunit.
• It is about 100 Angstroms long.
• It can just about accommodate the diameter of an α helix.
• The first third of it is made of RNA.
• Then the wall is composed of a mosaic of small hydrophobic and hydrophilic patches (i.e. non-stick).
• We don’t know if the peptide moves actively or passively.
• Antibiotics function in the exit channel to inhibit translation.
What does paromycin do?
- Paromycin works by reducing the accuracy of the process.
- It favours G530 and A1492/1493 in the ON conformation.
- This means that 30S domain closure is favoured and GTPase activation is accelerated no matter if the tRNA is cognate or near cognate.
How can antibiotics be used to study translocation?
- Fusidic acid allows GTP hydrolysis but it prevents EF-G turnover.
- Thiostrepton allows GTP hydrolysis but prevents conformational change required to turnover EF-G. It acts before fusidic acid.
