Translation Arrest Requires Two-Way Communication between a Nascent Polypeptide and the Ribosome  Cheryl A. Woolhead, Arthur E. Johnson, Harris D. Bernstein  Molecular Cell  Volume 22, Issue 5, Pages 587-598 (June 2006) DOI: 10.1016/j.molcel.2006.05.021 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 SecM Derivatives Containing Fluorescent Probes Undergo Translation Arrest (A) The principal constructs used in this study are shown. The first 37 residues of SecM constitute a signal sequence (SS). The amino acids near the C terminus of SecM that are essential for translation arrest are underlined, and the point at which arrest occurs is indicated. Fluorescent probes were incorporated into unique amber and Lys codons in SecM DA. (B) The incorporation of fluorescent probes into SecM DA is schematized. The acceptor probe (○) is first incorporated at Amb 135 (UAG) using ɛBOP-Lys-tRNAamb, and the donor probe (●) is then incorporated at Lys 159 (AAA) residue using ɛBOP-Lys-tRNALys. (C) SecM was synthesized in a coupled transcription-translation reaction, and the reaction products were subjected to CTABr precipitation. Polypeptides present in the CTABr pellet (P) and supernatant (S) were resolved by SDS-PAGE. (D) SecM containing an amber codon at position 159 (SecM 159Amb) was synthesized in vitro in the presence of ɛBOP-[3H]Lys-tRNAamb, and the reaction products were subject to CTABr precipitation. (E) SecM DA was synthesized in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of ɛBOP-[3H]Lys-tRNAamb and ɛBOF-[14C]Lys-tRNALys, and the reaction products were subject to CTABr precipitation. Molecular Cell 2006 22, 587-598DOI: (10.1016/j.molcel.2006.05.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 The C Terminus of Nascent SecM Undergoes a Conformational Change upon Completion of the Arrest Motif (A) RNC complexes containing SecM DA (1–162), SecM DA (1–164), and SecM DA (1–166) were purified, and the efficiency of energy transfer (E) was determined. Error bars represent the standard deviation of E obtained in three or more independent experiments. The location of the donor (○) and acceptor (●) probes in each nascent chain is illustrated. (B) Illustration of an extended polypeptide conformation (top) and two types of compacted conformations (middle, bottom) that are compatible with the data. Molecular Cell 2006 22, 587-598DOI: (10.1016/j.molcel.2006.05.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Mutations that Prevent Compaction of the Nascent Chain Inhibit Translation Arrest (A) The SecM mutations that were analyzed in (B), (C), and (D) are shown. Amino acids that were previously shown to be essential for translation arrest are underlined. (B) The indicated SecM mutants were synthesized in a coupled transcription-translation reaction, and the reaction products were subjected to CTABr precipitation. Polypeptides present in the CTABr pellet (P) and supernatant (S) were resolved by SDS-PAGE. (C) RNC complexes containing the indicated derivatives of SecM DA (1–166) were purified, and the efficiency of energy transfer (E) was determined. Error bars represent the standard deviation of E obtained in three or more independent experiments. (D) DH5α transformed with pNH122 (SecM [121–166] − LacZα) containing either the wild-type (wt) SecM sequence or the indicated mutation were plated onto LB agar supplemented with 1 mM IPTG, 40 μg/ml X-gal, and 40 μg/ml chloramphenicol and grown at 37°C. Molecular Cell 2006 22, 587-598DOI: (10.1016/j.molcel.2006.05.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Compaction Is Induced by the Ribosome Tunnel (A) Wild-type SecM (wt) or a P153A mutant was synthesized in cell-free extracts derived from strains that harbor a deletion of amino acids 82–84 or a K63E mutation in ribosomal proteins L22 and L4, respectively. The reaction products were subjected to CTABr precipitation. Polypeptides present in the CTABr pellet (P) and supernatant (S) were resolved by SDS-PAGE. (B) Wild-type SecM DA (1–166) or the P153A mutant was synthesized in an extract derived from the strain that harbors a deletion in L22. RNC complexes were purified, and the efficiency of energy transfer (E) was determined. Error bars represent the standard deviation of E obtained in three or more independent experiments. Molecular Cell 2006 22, 587-598DOI: (10.1016/j.molcel.2006.05.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Mutation of Key Residues Impairs Translation Arrest But Does Not Affect the Folding of the Nascent Chain (A) The SecM mutations that were analyzed in (B) and (C) are shown. Amino acids that were previously shown to be essential for translation arrest are underlined. (B) The indicated SecM mutants were synthesized in a coupled transcription-translation reaction, and the reaction products were subjected to CTABr precipitation. Polypeptides present in the CTABr pellet (P) and supernatant (S) were resolved by SDS-PAGE. (C) RNC complexes containing the indicated derivatives of SecM DA (1–166) were purified, and the efficiency of energy transfer (E) was determined. Error bars represent the standard deviation of E obtained in three or more independent experiments. Molecular Cell 2006 22, 587-598DOI: (10.1016/j.molcel.2006.05.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 The Ribosome Undergoes Functional and Structural Changes during Translation Arrest (A) (Left panel) Purified RNC complexes containing SecM (1–162), SecM (1–164), or SecM (1–166) were either mock treated (lanes 1–3) or treated with RNase A (lanes 4–6). (Right panel) Purified RNC complexes containing SecM (1–153) or SecM (1–153) with a P153A mutation were either mock treated (lanes 1 and 3) or treated with RNase A (lanes 2 and 4). All samples were analyzed by SDS-PAGE. (B) Wild-type SecM (1–166) (wt) or the indicated mutant was synthesized in a coupled transcription-translation reaction and incubated with 0 mM, 0.5 mM, or 5 mM puromycin prior to SDS-PAGE. (C) Purified RNC complexes containing wild-type SecM K (1–166) (wt) or the indicated mutant were untreated (top panel), incubated with 1 mM DSS (middle panel), or subjected to immunoprecipitation with an anti-L23 antiserum after treatment with 1 mM DSS (bottom panel) and then analyzed by SDS-PAGE. Molecular Cell 2006 22, 587-598DOI: (10.1016/j.molcel.2006.05.021) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 A Model for the Mechanism of Translation Arrest The SecM nascent chain is in an extended conformation (1) until synthesis of the arrest motif is completed. At that point, the ribosome induces the C terminus of the nascent chain to adopt a more compact structure (2). This conformational change orients key residues in SecM (★) so as to facilitate their interaction with ribosomal components that line the tunnel (3). This interaction ultimately leads to a further change in the conformation or status of the RNC complex that in turn arrests polypeptide elongation. Molecular Cell 2006 22, 587-598DOI: (10.1016/j.molcel.2006.05.021) Copyright © 2006 Elsevier Inc. Terms and Conditions