A cryo-electron microscopic study of ribosome-bound termination factor RF2

Rawat, U.; Zavialov, Andrey V.; Sengupta, Jayati; Valle, Mikel; Grassucci, Robert A.; Linde, Jamie; Vestergaard, Bente; Ehrenberg, Måns; Frank, Joachim

doi:10.1142/9789813234864_0031

Cited by 14 publications

(21 citation statements)

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“…In the pre-termination (pre-hydrolysis) state, the substrate peptidyl-tRNA resides in a nonrotated (classical) conformation of 70S ribosome, with its anticodon stem loop bound to the mRNA codon in the P site of the small 30S subunit and the CCA-peptidyl moiety in the P site of the large 50S subunit Ermolenko et al, 2007). Cryogenic electron microscopy (cryo-EM) and X-ray studies reveal RF1 or RF2 bound in the A site next to P tRNA (Klaholz et al, 2003;Petry et al, 2005;Rawat et al, 2006;Rawat et al, 2003), and provide insight into pre-hydrolysis (Jin et al, 2010) and post-hydrolysis states Korostelev et al, 2010;Laurberg et al, 2008;Weixlbaumer et al, 2008). The release factor's codon-recognition domain binds the stop codon in the 30S decoding center.…”

Section: Introductionmentioning

confidence: 99%

Extensive Ribosome and RF2 Rearrangements during Translation Termination

Svidritskiy

Demo

Loveland

et al. 2019

Preprint

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Protein synthesis ends when a ribosome reaches an mRNA stop codon. Release factors (RFs) decode the stop codon, hydrolyze peptidyl-tRNA to release the nascent protein, and then dissociate to allow ribosome recycling. To visualize termination by RF2, we resolved a cryo-EM ensemble of E. coli 70S•RF2 structures at up to 3.3 Å in a single sample. Five structures suggest a highly dynamic termination pathway. Upon peptidyl-tRNA hydrolysis, the CCA end of deacyl-tRNA departs from the peptidyl transferase center. The catalytic GGQ loop of RF2 is rearranged into a long -hairpin that plugs the peptide tunnel, biasing a nascent protein toward the ribosome exit. Ribosomal intersubunit rotation destabilizes the catalytic RF2 domain on the 50S subunit and disassembles the central intersubunit bridge B2a, resulting in RF2 departure. Our structures visualize how local rearrangements and spontaneous inter-subunit rotation poise the newly-made protein and RF2 to dissociate in preparation for ribosome recycling.

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Section: Introductionmentioning

confidence: 99%

Extensive Ribosome and RF2 Rearrangements during Translation Termination

Svidritskiy

Demo

Loveland

et al. 2019

Preprint

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“…The high sequence similarity between RF1 and RF2 suggests that the two factors interact with the ribosome in the same manner and 6 promote peptide release by a similar mechanism (Freistroffer et al, 1997;Zavialov et al, 2001). However, structures of RF1 or RF2 bound to PostHC reveal differences regarding the interaction with the L11 region of the 50S subunit Laurberg et al, 2008;Petry et al, 2005;Pierson et al, 2016;Rawat et al, 2006;Rawat et al, 2003;Weixlbaumer et al, 2008). Thus, it is not clear whether RF1 and RF2 follow the same mechanism and whether they respond in the same way to the recruitment of RF3 to termination complexes.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of ribosomes and release factors during translation termination inE. coli

Adio

Sharma

Senyushkina

et al. 2018

Preprint

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Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination.The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET together with ensemble kinetics, we show that ribosome termination complexes that carry two factors, RF1-RF3 or RF2-RF3, are dynamic and fluctuate between non-rotated and rotated states, while each factor alone has its distinct signature on the ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. GTP cleavage helps RF3 release from ribosomes stalled in the rotated state in the absence of RF1. Our data suggest how the stochastic assembly of the ribosome-RF1-RF3-GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

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“…One model to explain the accuracy of translation termination is that stop-codon recognition triggers a large-scale conformational rearrangement that activates release factor (11)(12)(13)(14). Many structural studies of 70S ribosome termination complexes formed with RF1 or RF2 (11,12,(15)(16)(17)(18)(19)(20)(21) have revealed an active, i.e. extended, conformation of release factor.…”

Section: Introductionmentioning

confidence: 99%

Conformational control of translation termination on the 70S ribosome

Svidritskiy¹,

Коростелев²

2017

Preprint

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Translation termination ensures proper lengths of cellular proteins. During termination, release factor (RF) recognizes a stop codon and catalyzes peptide release.Conformational changes in RF are thought to underlie accurate translation termination.If true, the release factor should bind the A-site codon in inactive (compact) conformation(s), but structural studies of ribosome termination complexes have only captured RFs in an extended, active conformation. Here, we identify a hyper-accurate RF1 variant, and present crystal structures of 70S termination complexes that suggest a structural pathway for RF1 activation. In the presence of blasticidin S, the catalytic domain of RF1 is removed from the peptidyl-transferase center, whereas the codonrecognition domain is fully engaged in stop-codon recognition in the decoding center.RF1 codon recognition induces decoding-center rearrangements that precede accommodation of the catalytic domain. Our findings suggest how structural dynamics of RF1 and the ribosome coordinate stop-codon recognition with peptide release, ensuring accurate translation termination.

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A cryo-electron microscopic study of ribosome-bound termination factor RF2

Cited by 14 publications

References 0 publications

Extensive Ribosome and RF2 Rearrangements during Translation Termination

Extensive Ribosome and RF2 Rearrangements during Translation Termination

Dynamics of ribosomes and release factors during translation termination inE. coli

Conformational control of translation termination on the 70S ribosome

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