The Plasticity of a Translation Arrest Motif Yields Insights into Nascent Polypeptide Recognition inside the Ribosome Tunnel

Yap, Mee-Ngan Frances; Bernstein, Harris

doi:10.1016/j.molcel.2009.04.002

Cited by 99 publications

(125 citation statements)

References 35 publications

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“…(B) Diversity in the intrinsic class of arrest sequences. Shown are the amino acid sequences that induce translation arrest without any cofactor, which have been elucidated for VemP, SecM (18,52), and MifM (19). Residues whose identities contribute to the elongation arrest are highlighted in blue; particularly important residues are shown in boldface.…”

Section: Discussionmentioning

confidence: 99%

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

Ishii

Chiba

Hashimoto

et al. 2015

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

112

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(A) Predicted secondary structure of mRNA at the vemP-secD2 VA intergenic region. The RNA sequence from the fifth last codon of vemP to the third codon of secD2 VA are shown with the secondary structure predicted by CentroidHomfold. The putative SD sequence and the start codon of secD2 VA are indicated by box and underline, respectively. The P site and the A site of the VemP-stalled ribosome (Fig. 4C) are shown schematically. To separate the VemP arrest point and the secondary structure-forming region, we inserted one or two copies of the 18 nucleotides encoding the last five amino acid residues of VemP followed by the termination codon, shown at the bottom. (B) Separation of the arrest point and the stem-loop-forming region impairs the regulation. E. coli cells carrying indicated vemP-secDF2 VA plasmids (with or without the insertion mutations shown in A) were induced with IPTG for 15 min at 37°C and treated with (+) or without (−) 3 mM NaN 3 for 5 min. Cells were then pulse-labeled for 1 min. Cell extracts of equivalent radioactivities were subjected to anti-VemP (upper gel) and anti-V. SecD2 (lower gel) immunoprecipitation. In the upper gel, "p" indicates the signal peptide unprocessed form of VemP, which included the polypeptide part of the elongation arrested VemP, whereas "m" indicates the signal peptide-processed mature form. Relative intensities of V.SecD2 are shown in the bottom graph, taking the value of lane 1 as unity (n = 3). Gray and black bars represent results obtained without and with NaN 3 treatment, respectively. (C) The insertion mutations do not affect translation arrest of VemP. E. coli cells carrying pBAD24-Δss-vemP-V.secDF2 with or without the insertion mutations were induced with 0.02% arabinose for 1 h at 37°C. The same amounts of proteins were separated by 10% wide-range gel electrophoresis, followed by immunodetection of VemP. (D) The secD2 VA -secF2 VA interval. The nucleotide sequence from the fourth last codon of secD2 VA to the fifth codon of secF2 VA is shown. The predicted SD sequence of secF2 VA is underlined.

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Section: Discussionmentioning

confidence: 99%

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

Ishii

Chiba

Hashimoto

et al. 2015

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

112

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“…Finally, we note that the 10 C-terminal residues in the E. coli SecM AP (SQAQGIRAGP) differ from the Sec-M(Ms-Sup1) AP by sequence changes that, according to the data in Fig. 2, should make it less potent, as indeed is the case (13,18). The recent discovery that oligo-proline stretches stall translation in the absence of EF-P (3,20) prompted us to investigate whether this kind of stalling also could be overcome by pulling on the nascent chain.…”

Section: Discussionmentioning

confidence: 76%

“…To screen for APs that can withstand strong pulling forces, we used a previously identified mutant version of the M. succiniciproducens SecM AP, called SecM(Ms-Sup1) (13,16,18), as the starting template. The AP was inserted close to the C terminus of the E. coli inner membrane protein leader peptidase (LepB) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For the SecM(Ms-Sup1) AP, we substituted every amino acid for every other amino acid in the sequence SSHPPIRGSPGS (where the underlined segment is the AP as defined by Ala scanning (18)) and analyzed the arrest potency under a rather strong pulling force. In positions Ϫ7 to Ϫ1, only two mutations were found to increase the strength of the AP, namely Ser Ϫ2 3 Pro and Pro Ϫ6 3 Arg (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Aromatic residues in position Ϫ7 (Tyr) and Ϫ10 (Phe) also have counterparts in the SecM(Ms-Sup1) AP mutational screen. A third study (18) identified revertants of a weakened variant of the E. coli SecM AP. Again, the results agree with ours (in particular, the strong stalling potency of a revertant AP with cysteine in position Ϫ10 was evident in this study, cf.…”

Section: Discussionmentioning

confidence: 99%

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Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Cymer

Hedman

Ismail

et al. 2015

Journal of Biological Chemistry

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Background:The stalling efficiency of translational arrest peptides (APs) is sensitive to mechanical pulling forces on the nascent chain. Results: We identify new APs with enhanced stalling efficiency. Conclusion: Mechanical pulling forces reduce stalling induced by diproline stretches in efp Ϫ cells. Significance:Our results provide new insights into AP-induced translational stalling and offer new in vivo force sensors.Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.During protein synthesis, the nascent polypeptide chain moves through the ϳ100-Å-long exit tunnel in the large ribosomal subunit (1). Strong interactions between the nascent chain and the tunnel might adversely affect protein synthesis and can even lead to complete blockage of the ribosome, a mechanism exploited by many antibiotics (2). It has therefore been postulated that the ribosome exit tunnel has a "Teflonlike" surface that minimizes interactions with the nascent chain to avoid adverse effects on translation (1, 3).Nevertheless, some nascent chain segments are able to interact with the ribosomal exit tunnel in ways that block or slow down translation (4, 5). In bacteria, such translational arrest peptides (APs) 2 are often used to regulate the translation of downstream open reading frames in polycistronic mRNAs (6, 7). APs interact with distinct ribosomal RNA and protein components within the ribosomal exit tunnel (8), inducing conformations at the ribosome active site that can block the peptidyl transfer reaction (9 -11).In the case of the AP-containing SecM protein in Escherichia coli, the arrest of nascent chain elongation can be overcome by the activity of the motor protein SecA, which presumably breaks the AP-tunnel interactions by mechanically pulling on the nascent chain (12). On the basis of this notion, we hypothesized that...

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Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

et al. 2022

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During SecYEG‐mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N‐terminal end is ~ 45 residues away from the peptidyl transferase centre. However, we recently uncovered instances where the first contact is delayed by up to ~ 10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the Escherichia coli inner membrane protein BtuC: a positively charged loop and a re‐entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re‐entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.

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The Plasticity of a Translation Arrest Motif Yields Insights into Nascent Polypeptide Recognition inside the Ribosome Tunnel

Cited by 99 publications

References 35 publications

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

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