Elongation Arrest by SecM via a Cascade of Ribosomal RNA Rearrangements

Mitra, Kakoli; Schaffitzel, Christiane; Fabiola, Felcy; Chapman, Michael S.; Ban, Nenad; Frank, Joachim

doi:10.1016/j.molcel.2006.05.003

Cited by 76 publications

(82 citation statements)

References 34 publications

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“…Previous reports have proposed that interaction between SecM and nucleotide A2058 of 23S rRNA is important for SecM-mediated pausing (20,21). While the SecM peptide may interact with A2058, as suggested by the effect of the A2058G mutation, any putative interaction does not mimic the interaction of erythromycin with A2058, since we showed that methylation of A2058 by the ErmC methylase inhibits the binding of erythromycin but has no affect on SecM activity.…”

Section: Discussionmentioning

confidence: 45%

Effects on Translation Pausing of Alterations in Protein and RNA Components of the Ribosome Exit Tunnel

2008

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Amino acids are polymerized into peptides in the peptidyl transferase center of the ribosome. The nascent peptides then pass through the exit tunnel before they reach the extraribosomal environment. A number of nascent peptides interact with the exit tunnel and stall elongation at specific sites within their peptide chain. Several mutational changes in RNA and protein components of the ribosome have previously been shown to interfere with pausing. These changes are localized in the narrowest region of the tunnel, near a constriction formed by ribosomal proteins L4 and L22. To expand our knowledge about peptide-induced pausing, we performed a comparative study of pausing induced by two peptides, SecM and a short peptide, CrbCmlA , that requires chloramphenicol as a coinducer of pausing. We analyzed the effects of 15 mutational changes in L4 and L22, as well as the effects of methylating nucleotide A2058 of 23S rRNA, a nucleotide previously implicated in pausing and located close to the L4-L22 constriction. Our results show that methylation of A2058 and most mutational changes in L4 and L22 have differential effects on pausing in response to Crb CmlA and SecM. Only one change, a 6-amino-acid insertion after amino acid 72 in L4, affects pausing in both peptides. We conclude that the two peptides interact with different regions of the exit tunnel. Our results suggest that either the two peptides use different mechanisms of pausing or they interact differently but induce similar inhibitory conformational changes in functionally important regions of the ribosome.Peptide bond formation during translation takes place within the large subunit of the ribosome, at the peptidyl transferase center (PTC). In order to escape the ribosome, newly formed peptides must traverse a 100-Å-long channel referred to as the peptide exit tunnel. This tunnel is lined primarily by segments of 23S rRNA, although two ribosomal proteins, L4 and L22, also contribute part of the tunnel lining (1, 26). These two r-protein components form a constriction that results in the narrowest passage in the tunnel (Fig. 1). Mutational changes in both rRNA and r-protein components located near the constriction are important for bacterial resistance to macrolide-lincosamide-streptogramin antibiotics. These antibiotics bind to nucleotides A2058 and 2059 and other close-by portions of 23S rRNA (reviewed in reference 11).The tunnel constriction has also been implicated in peptidemediated pausing (7, 21). Peptide-mediated pausing occurs when sequence-specific interactions between the nascent peptide and the translating ribosome cause a stall in translation. SecM is a well-characterized example of a pause-inducing peptide. Mutations affecting the efficiency of SecM-induced pausing are found in the codons for a 17-amino-acid stretch of the peptide, termed the pause motif (21). This region is sufficient for inducing pausing when inserted into another protein sequence (10, 21). The SecM pausing response is also decreased by mutations altering the ribosome. Two of the...

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Section: Discussionmentioning

confidence: 45%

Effects on Translation Pausing of Alterations in Protein and RNA Components of the Ribosome Exit Tunnel

2008

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“…The 70S peak then completely disappears at 3.0 M urea, and the 50S peak broadens and simultaneously decreases in intensity, signifying denaturation of 50S subunits. SecM-stalled ribosomes should be more stable than reconstituted 70S ribosomes because the SecM stalling sequence is known to make extensive contacts with the ribosome exit tunnel and to strengthen 30S-50S contacts (19). To test the urea sensitivity of SecM-stalled RNCs, we translated DHFR V75R-(GS) 5 -SecM for 1 h, halted translation with 2 mM chloramphenicol, incubated the IVT reactions in urea overnight, and subjected these samples to sucrose-gradient ultracentrifugation.…”

Section: Resultsmentioning

confidence: 99%

Quantitative determination of ribosome nascent chain stability

Samelson

Jensen

Soto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

131

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Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.protein folding | cotranslational folding | pulse proteolysis | protein stability P roper protein folding is necessary for the function of all cells, and changes in cellular protein folding capacity can lead to cell death and disease (1). For more than 50 years, experimental studies have probed the physical properties of proteins, paving the way for incredible advances in protein design and protein structure prediction, as well as for understanding the first principles of protein folding (2, 3). These in vitro studies, however, do not necessarily recapitulate the folding process in vivo, including the constraints that the ribosome imposes on the emerging nascent chain during translation (4).In the cell, proteins are synthesized by the ribosome one amino acid at a time on a time scale that is slower than most in vitro protein folding rates (5). Thus, the emerging chain has time to explore conformational space and adopt structured conformations before its entire sequence has been synthesized (6). This vectorial process, and the proximity of the ribosome itself, can modulate the emerging chain's energy landscape in ways that are just beginning to be appreciated. Translation can affect folding efficiency, enable the population of intermediates that are not revealed during offribosome studies, and even determine the final conformational fate of the nascent protein (6-10). To understand the folding process in vivo, general rules and biophysical mechanisms for these modulations of the emerging chain's energy landscape need to be elucidated.To unravel the in vivo folding landscape, we need to interrogate the energetics and dynamics of ribosome-bound nascent chains in a manner analogous to studies of the in vitro folding process. The challenge, however, is that standard ...

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“…Thus, one may speculate that the signal leading to inhibition of peptidyl transfer upon synthesis of SecM at least in part may be transmitted through a 23S rRNAmediated pathway that would target U2506. Based on cryo-EM data, a model for the transmission of the SecM signal toward the PT center via a cascade of rRNA rearrangements has been proposed (Mitra et al 2006). Nucleotides in the PT center itself have not been identified as suppressors, most likely because mutations of most of these residues confer lethal phenotype.…”

Section: Modulation Of Peptidyl Transferase Activitymentioning

confidence: 99%

Modulating the activity of the peptidyl transferase center of the ribosome

Beringer¹

2008

RNA

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The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center.

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Elongation Arrest by SecM via a Cascade of Ribosomal RNA Rearrangements

Cited by 76 publications

References 34 publications

Effects on Translation Pausing of Alterations in Protein and RNA Components of the Ribosome Exit Tunnel

Effects on Translation Pausing of Alterations in Protein and RNA Components of the Ribosome Exit Tunnel

Quantitative determination of ribosome nascent chain stability

Modulating the activity of the peptidyl transferase center of the ribosome

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