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: 14.46). 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: All proteins, from bacteria to man, are made in the ribosome and are elongated, one residue at a time, at the peptidyl transferase center (PTC). This growing peptide chain wends its way through the ribosomal tunnel to the exit port, ~ 100 angstroms from the PTC. We have identified locations in the tunnel that sense and respond to single side chains of the nascent peptide to induce local conformational changes. Moreover, side-chain sterics and rearrangements deep in the tunnel influence the disposition of residues 45Å away at the exit port and are consistent with side-chain induced axial retraction of the peptide backbone. These coupled responses are neither haphazard nor uniform along the tunnel. Rather, they are confined to discriminating zones in the tunnel and are sequence-specific. Such discerning communication may contribute to folding events and mechanisms governing sequence-specific signaling between different regions of the tunnel during translation.
    Journal of Molecular Biology 10/2014; 426(24). DOI:10.1016/j.jmb.2014.10.006 · 3.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47-49 residues away from the ribosomal P site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential, whereas the second may reflect interactions between charged residues and the periplasmic membrane surface.
    Nature Structural & Molecular Biology 01/2015; DOI:10.1038/nsmb.2940 · 11.63 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Upstream open reading frames (uORFs) are often found in the 5'-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 01/2015; 43(3). DOI:10.1093/nar/gkv018 · 8.81 Impact Factor

Full-text (2 Sources)

Available from
Feb 19, 2015