When the mRNA translating ribosome encounters a stop codon in its aminoacyl site (A site), it recruits a class-1 release factor (RF) to induce hydrolysis of the ester bond between peptide chain and peptidyl-site (P-site) tRNA. This process, called termination of translation, is under strong selection pressure for high speed and accuracy. Class-1 RFs (RF1, RF2 in bacteria, eRF1 in eukarya and aRF1 in archaea), have structural motifs that recognize stop codons in the decoding center (DC) and a universal GGQ motif for induction of ester bond hydrolysis in the peptidyl transfer center (PTC) 70 Å away from the DC. The finding that free RF2 is compact with only 20 Å between its codon reading and GGQ motifs came therefore as a surprise. Cryo-electron microscopy (cryo-EM) then showed that ribosome-bound RF1 and RF2 have extended structures, suggesting that bacterial RFs are compact when entering the ribosome and switch to the extended form in a stop signal-dependent manner. FRET, cryo-EM and X-ray crystallography, along with a rapid kinetics study suggesting a pre-termination conformational change on the millisecond time-scale of ribosome-bound RF1 and RF2, have lent indirect support to this proposal. However, direct experimental evidence for such a short-lived compact conformation on the native pathway to RF-dependent termination is missing due to its transient nature. Here we use time-resolved cryo-EM to visualize compact and extended forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing complex on the pathway to termination. About 25% of ribosomal complexes have RFs in the compact state at 24 ms reaction time after mixing RF and ribosomes, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.
Date: 25 February 2019, 11:00 (Monday, 7th week, Hilary 2019)
Venue: Wellcome Trust Centre for Human Genetics, Headington OX3 7BN
Venue Details: Meeting room A
Speaker: Dr Ziao Fu (Columbia University of New York)
Organising department: Wellcome Trust Centre for Human Genetics
Organiser: Agata Krupa (Wellcome Centre for Human Genetics, University of Oxford)
Host: Prof Peijun Zhang (University of Oxford )
Part of: Strubi seminars
Booking required?: Not required
Audience: Members of the University only
Editor: Agata Krupa