RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity
Martino Morici,
Sara Gabrielli,
Keigo Fujiwara
et al.
Abstract:Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent… Show more
“…However, the stalling efficiencies on E. coli and B. subtilis ribosomes varied among each arrest peptide (Figs. 2 , 4 ), suggesting that the residues located upstream of the RAPP motif play an important role in determining the species specificity, as suggested for ApdA, and ApdP 28 , 36 . In addition, during the identification of YwcI, we found that most of the E. coli and B. subtilis proteins that contained RAPP-like motifs did not stall the ribosome (Supplementary Fig.…”
Section: Discussionmentioning
confidence: 84%
“…3c, d ). Previous studies demonstrated that the stalling of ApcA, ApdA, and ApdP also occurred at the third position of the RAPG/RAPP sequences, suggesting that these arrest peptides share a common underlying mechanism 36 . Fig.…”
Section: Resultsmentioning
confidence: 96%
“…The sharing of a similar RAPP-like sequence makes it plausible that these arrest peptides employ a similar nascent chain-ribosome interaction near the PTC to achieve ribosome stalling, as suggested by structural studies of ApdA, ApdP, and SecM 36 , 49 . This might also be the case for the arrest-inducing sequences containing RAPP-like motifs that were identified in a screening using a random sequence library 50 , 51 .…”
Regulatory arrest peptides interact with specific residues on bacterial ribosomes and arrest their own translation. Here, we analyse over 30,000 bacterial genome sequences to identify additional Sec/YidC-related arrest peptides, followed by in vivo and in vitro analyses. We find that Sec/YidC-related arrest peptides show patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Several of the identified peptides contain distinct conserved sequences near the C-termini, but are still able to efficiently stall bacterial ribosomes in vitro and in vivo. In addition, we identify many arrest peptides that share an R-A-P-P-like sequence, suggesting that this sequence might serve as a common evolutionary seed to overcome ribosomal structural differences across species.
“…However, the stalling efficiencies on E. coli and B. subtilis ribosomes varied among each arrest peptide (Figs. 2 , 4 ), suggesting that the residues located upstream of the RAPP motif play an important role in determining the species specificity, as suggested for ApdA, and ApdP 28 , 36 . In addition, during the identification of YwcI, we found that most of the E. coli and B. subtilis proteins that contained RAPP-like motifs did not stall the ribosome (Supplementary Fig.…”
Section: Discussionmentioning
confidence: 84%
“…3c, d ). Previous studies demonstrated that the stalling of ApcA, ApdA, and ApdP also occurred at the third position of the RAPG/RAPP sequences, suggesting that these arrest peptides share a common underlying mechanism 36 . Fig.…”
Section: Resultsmentioning
confidence: 96%
“…The sharing of a similar RAPP-like sequence makes it plausible that these arrest peptides employ a similar nascent chain-ribosome interaction near the PTC to achieve ribosome stalling, as suggested by structural studies of ApdA, ApdP, and SecM 36 , 49 . This might also be the case for the arrest-inducing sequences containing RAPP-like motifs that were identified in a screening using a random sequence library 50 , 51 .…”
Regulatory arrest peptides interact with specific residues on bacterial ribosomes and arrest their own translation. Here, we analyse over 30,000 bacterial genome sequences to identify additional Sec/YidC-related arrest peptides, followed by in vivo and in vitro analyses. We find that Sec/YidC-related arrest peptides show patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Several of the identified peptides contain distinct conserved sequences near the C-termini, but are still able to efficiently stall bacterial ribosomes in vitro and in vivo. In addition, we identify many arrest peptides that share an R-A-P-P-like sequence, suggesting that this sequence might serve as a common evolutionary seed to overcome ribosomal structural differences across species.
Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.
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