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The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

Ziao Fu, Gabriele Indrisiunaite, Sandip Kaledhonkar, Binita Shah, Ming Sun, Bo Chen, Robert A. Grassucci, Måns Ehrenberg, Joachim Frank
doi: https://doi.org/10.1101/470047
Ziao Fu
1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA
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Gabriele Indrisiunaite
2Department of Cell and Molecular Biology, Uppsala University, Sweden
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Sandip Kaledhonkar
1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA
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Binita Shah
3Department of Biological Sciences, Barnard College, New York, USA
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Ming Sun
4Department of Biological Sciences, Columbia University, New York, USA
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Bo Chen
4Department of Biological Sciences, Columbia University, New York, USA
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Robert A. Grassucci
1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA
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Måns Ehrenberg
2Department of Cell and Molecular Biology, Uppsala University, Sweden
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Joachim Frank
1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, USA
4Department of Biological Sciences, Columbia University, New York, USA
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Abstract

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 surprise1. Cryo-electron microscopy (cryo-EM) then showed that ribosome-bound RF1 and RF2 have extended structures2,3, suggesting that bacterial RFs are compact when entering the ribosome and switch to the extended form in a stop signal-dependent manner3. FRET4, cryo-EM5,6 and X-ray crystallography7, along with a rapid kinetics study suggesting a pre-termination conformational change on the millisecond time-scale of ribosome-bound RF1 and RF28, 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-EM9,10,11,12,13 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.

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Posted November 14, 2018.
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The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy
Ziao Fu, Gabriele Indrisiunaite, Sandip Kaledhonkar, Binita Shah, Ming Sun, Bo Chen, Robert A. Grassucci, Måns Ehrenberg, Joachim Frank
bioRxiv 470047; doi: https://doi.org/10.1101/470047
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The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy
Ziao Fu, Gabriele Indrisiunaite, Sandip Kaledhonkar, Binita Shah, Ming Sun, Bo Chen, Robert A. Grassucci, Måns Ehrenberg, Joachim Frank
bioRxiv 470047; doi: https://doi.org/10.1101/470047

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