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

Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Molecular cell (Impact Factor: 14.02). 05/2009; 34(2):201-11. DOI: 10.1016/j.molcel.2009.04.002
Source: PubMed

ABSTRACT The recognition of a C-terminal motif in E. coli SecM ((150)FXXXXWIXXXXGIRAGP(166)) inside the ribosome tunnel causes translation arrest, but the mechanism of recognition is unknown. Whereas single mutations in this motif impair recognition, we demonstrate that new arrest-inducing peptides can be created through remodeling of the SecM C terminus. We found that R163 is indispensable but that flanking residues that vary in number and position play an important secondary role in translation arrest. The observation that individual SecM variants showed a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on the results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intratunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the tunnel and help to explain the striking diversity of arrest-inducing peptides found throughout nature.

Download full-text


Available from: Mee-Ngan F Yap, Feb 19, 2015
29 Reads
  • Source
    • "Cloning and Purification of ADR1a-SecM (Ms-Sup1; L = 25) RNCs The E. coli SecM stalling sequence in the ADR1a-SecM (L = 25) construct was modified by mutating five residues to obtain the Sup1 version of the Mannheimia succiniciproducens SecM AP (HPPIRGSP) (Yap and Bernstein, 2009), yielding ADR1a-SecM (Ms-Sup1; L = 25). The construct was subsequently cloned into the p7XNH vector. "
    [Show abstract] [Hide abstract]
    ABSTRACT: At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 09/2015; 12(1-8). DOI:10.1016/j.celrep.2015.07.065 · 8.36 Impact Factor
  • Source
    • "Stall sequences within nascent chains dramatically alter elongation, leading to a prolonged arrest of translation and controlling expression of cotranscribed genes (Ito and Chiba, 2013; Oliver et al., 1998). The SecM stall sequence from Escherichia coli relies solely on peptide-ribosome interactions to stall elongation (Nakatogawa and Ito, 2001; Yap and Bernstein, 2009). In secretion-deficient conditions, SecM-induced stalling upregulates SecA expression , an ATPase secretion protein (McNicholas et al., 1997; Schmidt et al., 1988; Yap and Bernstein, 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within 1 min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation.
    Cell Reports 05/2014; 7(5). DOI:10.1016/j.celrep.2014.04.033 · 8.36 Impact Factor
  • Source
    • ", 2009 ; Vazquez - Laslop et al . , 2008 ; Yap and Bernstein , 2009 ) . Interaction of a stalling polypeptide with the ribosome near or at the PTC could be a straightforward way to inhibit the catalytic activity . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus subtilis MifM uses polypeptide-instructed ribosomal stalling to control translation of YidC2, a membrane protein biogenesis factor. In contrast to other stalling systems involving a single arrest point, our in vitro translation/toeprint experiments show that the B. subtilis ribosome stalls consecutively at multiple codons of MifM. This mode of elongation arrest depends on nascent chain residues at the middle of the ribosomal exit tunnel and a few (four for the maximum functionality) negative charges residing proximally to the arrest points. The latter element does not require exact amino acid sequence, and this feature may underlie the multisite stalling. The arrested nascent chains were not efficiently transferred to puromycin, suggesting that growing MifM nascent chains inhibit peptidyl transferase center after acquiring an acidic residue(s). Multisite stalling seems to provide a unique means for MifM to achieve a sufficient duration of ribosomal stalling required for the regulatory function.
    Molecular cell 08/2012; 47(6):863-72. DOI:10.1016/j.molcel.2012.06.034 · 14.02 Impact Factor
Show more