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Visualization of codon-dependent conformational rearrangements during translation termination.

ABSTRACT: Although the recognition of stop codons by class 1 release factors (RFs) on the ribosome takes place with extremely high fidelity, the molecular mechanisms behind this remarkable process are poorly understood. Here we performed structural probing experiments with Fe(II)-derivatized RFs to compare the conformations of cognate and near-cognate ribosome termination complexes. The structural differences that we document provide an unprecedented view of how authentic stop-codon recognition is signaled to the large subunit of the ribosome. These events initiate with very close interactions between RF and the small-subunit decoding center, lead to increased interactions between the switch loop of the RF and specific regions of the subunit interface and end in the adoption of the precise orientation of the RF for maximal catalytic activity in the large-subunit peptidyl transferase center.

SUBMITTER: He SL

PROVIDER: S-EPMC2853735 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Visualization of codon-dependent conformational rearrangements during translation termination.

He Shan L SL Green Rachel R

Nature structural & molecular biology 20100307 4

Although the recognition of stop codons by class 1 release factors (RFs) on the ribosome takes place with extremely high fidelity, the molecular mechanisms behind this remarkable process are poorly understood. Here we performed structural probing experiments with Fe(II)-derivatized RFs to compare the conformations of cognate and near-cognate ribosome termination complexes. The structural differences that we document provide an unprecedented view of how authentic stop-codon recognition is signale ...[more]

PMID: 20208546

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Extensive ribosome and RF2 rearrangements during translation termination.

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Conformational Control of Translation Termination on the 70S Ribosome.

Project description: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. However, structural studies of ribosome termination complexes have only captured RFs in a conformation that is consistent with the catalytically active state. Here, we employ a hyper-accurate RF1 variant to obtain crystal structures of 70S termination complexes that suggest a structural pathway for RF1 activation. We trapped RF1 conformations with the catalytic domain outside of the peptidyl-transferase center, while the codon-recognition domain binds the stop codon. Stop-codon recognition induces 30S decoding-center rearrangements that precede accommodation of the catalytic domain. The separation of codon recognition from the opening of the catalytic domain suggests how rearrangements in RF1 and in the ribosomal decoding center coordinate stop-codon recognition with peptide release, ensuring accurate translation termination.

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Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1.

Project description:During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome.

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Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells.

Project description:Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined "initiation-specific" binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1? as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.

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