Lecture 4: Initiation and Termination Flashcards
How did we discover initiation factors?
- Initiation can be studied by translating R17 RNA.
- 70S ribosomes initiate translation.
- Washing with high salt buffer abolishes initiation.
- It can be restarted by adding soluble proteins purified from the high salt washings.
- Three proteins were isolated, the initiation factors: IF1, IF2 and IF3.
How do mRNA and tRNA-fMet bind?
Initiation begins with N.fmet and tRNA-fmet.
• This is highly specific. There is no binding to met-tRNA. The two tRNA species differ in sequence and tertiary structure. For example, tRNA-fmet has the anticodon of CAU while tRNA-mmet has an anticodon of N4-acetyl-CAU.
• The Shine-Dalgarno sequence is 5-12 bases 5’ of the AUG. The consensus sequence is complementary to the 3’ end of the 16S rRNA.
• mRNAs with secondary structures are bound more slowly. They can be unfolded with IF2-GTP. This is antagonised by IF3.
How do the initiation factors work?
The 3 initiation factors are IF1, IF2 and IF3.
• A vacant 30S ribosomal subunit binds IF3 and IF2.
• IF1 then binds. IF1 binds to the 30S ribosomal subunit in order to block the A site.
• Kinetic control of locking step is from IFs. IF2 is mainly responsible for increasing kon while IF3 strongly increases koff.
What does experimental evidence show us about the process of termination and how it links to elongation?
Termination is when the peptide chain is removed from the tRNA.
• Termination appears at the codons UAG, UAA or UGA.
• A simple model system used a start codon followed by a termination codon.
• It was found that the process required active factors to occur.
• The chain-terminating codon is not recognised by tRNA.
• The release factors can be purified. They are shown to release NfMet from tRNA in the assay.
• RF1 and RF2 bind to different codons.
• Some antibiotics inhibit transfer of the peptide and termination. However, certain mutations in 23S rRNA (50S subunit) abolish termination but not elongation. This implies that there are different catalytic mechanisms but they are both promoted by peptidyl transferase activity.
How do release factors work?
Release factors alter the specificity of the peptidyl transferase reaction.
• In termination, the ester linkage is transferred is to water in order to achieve hydrolysis.
• After release of peptide, RF1 and RF2 remain on the ribosome.
• RF3 is required to recycle them. RF3-GDP can bind to ribosome-RF1/2 complexes weakly. GTP then binds to the complex. This displaces RF1/2.
• Hydrolysis of GTP then liberates RF3-GDP. This leaves a complex of ribosome-mRNA-deacylated-tRNA. The complex is dissociated with ribosome releasing factor (RRF).
• The efficiency of termination depends on the nucleotide to the 3’ side of the termination codon. Termination is weak with A strong with U.
How does termination work at a chemical level?
Termination is catalysed by the PTC centre of the ribosome.
• The backbone NH group of Gln230 in the universally conserved GGQ motif is positioned so it can hydrogen bond with the 3’ OH of A76 of the peptidyl tRNA. The motif is in the release factors.
• 23S rRNA is involved in translation termination by RF2.
Which structural changes occur after mRNA and tRNA bind?
- The 30S subunit with initiation triplet in the P-site is docked by a 50S subunit.
- The GTPase function of IF2 is activated. GDP and Pi are left bound to IF2.
- IF3 NTD loses contact with the ribosome. Ribosomal affinity is reduced by 2 orders of magnitude.
- fMet-tRNA is adjusted on the ribosome, it occupies a P/I position which is intermediate between P/P and P/E.
- Pi dissociates from IF2-GDP. IF2 changes conformation. It loses contact with the acceptor end of fMet-tRNA.
- IF2 leaves the ribosome (or moves away from the A-site) and clears the way for EF-Tu binding.
- The EF-Tu-GTP-aa-tRNA complex binds to the 70S and through a number of steps delivers to the aa-tRNA to the ribosomal A-site. A peptide bond forms.
