The plasticity of a translation arrest motif yields insights into nascent polypeptide recognition inside the ribosome tunnel

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.

Fig. 1. Isolation of second-site mutations that suppress a defect in SecM-mediated translation arrest

(A) A plasmid encoding the translation arrest-deficient Q160P allele of SecM(Eco) was randomly mutagenized, and intragenic mutations that suppress the defect were isolated in a LacZα reporter screen. Positions where the sequence of the suppressor mutants (Sup1–6) and wild-type SecM differ are indicated. SecM(Eco) residues that were previously shown to be required for complete translation arrest are underlined. (B) CU164 (secY39) transformed with a plasmid encoding SecM(Eco), a SecM(Eco) mutant, or Sup1–6 were subjected to pulse-chase labeling after the addition of IPTG to induce expression of the plasmid-borne gene. Full-length and translation-arrested forms of SecM were immunoprecipitated and resolved by SDS-PAGE.

Fig. 2. C-terminal segments of distantly related SecM homologs induce translation arrest in E. coli

(A) Sequence alignment of the C-terminus of representative SecM homologs from Enterobacteriales (light gray) and Pastuerellales (dark gray). Invariant residues are indicated (●). Residues of SecM(Eco) that were previously shown to be required for full translation arrest are underlined. (B) Illustration of SecM(Eco) and SecM chimeras containing a C-terminal segment from H. influenzae 86-028NP SecM [SecM (C-Hi)] or M. succiniciproducens MBEL55E [SecM(C-Ms)]. Four residues were introduced after the arrest point (P166) to maintain a sequence length of 170 residues. Invariant, conserved and similar residues are depicted in black, dark grey and light grey, respectively. SS: signal sequence. (C) CU164 transformed with a plasmid encoding the indicated SecM derivative were subjected to pulse-chase labeling after the addition of IPTG, and SecM-containing polypeptides were immunoprecipitated.

Fig. 3. The translation of distantly related SecM variants stalls at the same position

The indicated SecM variants were synthesized in a coupled transcription-translation reaction and stalled peptidyl-tRNAs were analyzed by Northern blot using [γ-³²P]-ATP labeled oligonucleotides complementary to glyV/W/X/Y, proL, and serV tRNAs.

Fig. 4. The translation arrest motif in SecM (C-Ms) spans only eight residues

(A) CU164 transformed with a plasmid encoding SecM (C-Ms) or the indicated SecM (C-Ms) mutant were subjected to pulse-chase labeling after the addition of IPTG, and SecM-containing polypeptides were immunoprecipitated. (B) Summary of the data in part A and previous results (Nakatagowa and Ito, 2002).

Fig. 5. Prolines at positions 160 and 161 of SecM (Eco) reduce the role of F150, W155 and I156 in translation arrest

(A) and (B) CU164 transformed with a plasmid encoding SecM (Eco) or the indicated SecM (Eco) mutant were subjected to pulse-chase labeling after the addition of IPTG, and SecM-containing polypeptides were immunoprecipitated. The effect of combining prolines at positions 158 and 160 is illustrated in part B.

Fig. 6. SecM(Eco), Sup1 and SecM(C-Ms) nascent chains are located in distinct positions relative to ribosomal tunnel proteins

Photoreactive Bpa (A–D) or Azp (E–F) residues were incorporated into SecM(Eco), Sup1, and SecM(C-Ms) by amber suppression at the indicated position in coupled transcription-translation reactions. A portion of each sample was withdrawn for CTABr fractionation followed by immunoprecipitation with anti-SecM (lanes 1–6). P, CTABr pellet; S, CTABr supernatant. RNCs isolated from the remainder of each sample were subjected to immunoprecipitation with anti-SecM (lanes 7–9) or UV-irradiated and divided into portions that were subjected to immunoprecipitation with anti-SecM (lanes 10–12) or with anti-L4, L22 or L23 antisera (lanes 13–18). Prominent photoadducts are denoted with an arrowhead. In panel E, the asterisk denotes a nonspecific background band.

Fig. 7. Model for the recognition of SecM arrest peptides inside the ribosome tunnel

SecM-mediated translation arrest occurs when P166-tRNA is bound to the A site and the conformation adopted by the nascent chain inside the tunnel moves R163 into a precise intra-tunnel location. The presence of a proline at position 161 stabilizes the nascent chain conformation and effectively reduces the arrest peptide to the segment between residues 159–165. The presence of a flexible glycine at position 161, however, requires the stabilization of the nascent chain by residues farther down the tunnel (e.g., W155 and F150).