Multisite Ribosomal Stalling: A Unique Mode of Regulatory Nascent Chain Action Revealed for MifM  Shinobu Chiba, Koreaki Ito  Molecular Cell  Volume 47, Issue 6, Pages 863-872 (September 2012) DOI: 10.1016/j.molcel.2012.06.034 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 863-872DOI: (10.1016/j.molcel.2012.06.034) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Determination of the Sites of Elongation Arrest in mifM (A) Toeprint analysis. Coupled in vitro transcription-translation was directed with the mifM template using Bs PURE system (lanes 1 and 2) or that lacking the ribosome (lane 3) in the presence (lane 2) or the absence (lanes 1 and 3) of 0.1 mg/ml chloramphenicol (Cm). Translation complexes were then subjected to reverse transcription using a fluorescent primer followed by fluorimager visualization of the products. Lanes A, C, G, and T received respective dideoxy sequencing products. The numbers on the right represent nucleotide length of major toeprint products. (B) Assignment of the locations of the mifM-arrested ribosomes. In vitro transcription-translation, using Bs PURE system without including RF1, was directed with DNA templates of wild-type mifM (lanes 1 and 2) and the amber mutants (lanes 3–10) in the presence (even-numbered lanes) or the absence (odd-numbered lanes) of chloramphenicol. Samples were then toeprinted. Positions of the amber UAG codon are shown at the bottom. Numbers under “P” and “A” indicate the mifM codons in the P-site and the A-site, respectively, of the ribosomes stalled at an amber codon (open arrowheads) or at the MifM stalling sites (filled arrowheads). (C) Toeprint analysis of ermCL. Bs PURE system translation reactions were directed with the ermCL template in the presence (lane 1) or the absence (lane 2) of 10 μg/ml erythromycin and were then analyzed by toeprint assay. (D) Toeprint analysis of WPPP. Bs PURE system translation reactions were directed with the WPPP (lane 1) or the WPAP (lane 2) templates and were then analyzed by toeprint assay. (C) and (D) Lanes A, C, G, and T received respective dideoxy sequencing products. (E) Toeprint analysis of MifM variants with altered stalling abilities. Wild-type (lane 1) and the indicated mutants (lanes 2–6) of MifM were analyzed by in vitro translation and toeprinting. (F) Cartoon representation of the positions of the ribosome stalled on the mifM mRNA. Boxes “P” and “A” represent the residence of the P-site and the A-site of the ribosome, respectively, on the mRNA. Arrowheads indicate the last nucleotides of the reverse-transcription products observed upon toeprint assays. Molecular Cell 2012 47, 863-872DOI: (10.1016/j.molcel.2012.06.034) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Shift in the Arrest Population during the Early Time Course of Translation In vitro reaction to translate mifM mRNA was carried out with (lanes 1–5) and without (lanes 6–10) the B. subtilis ribosome for 2 (lanes 1 and 6), 4 (lanes 2 and 7), 8 (lanes 3 and 8), 16 (lanes 4 and 9), and 32 (lanes 5 and 10) min. Translation mixtures were then subjected to reverse transcription at 37°C for 2 min for toeprinting (right panel). Band intensities, from which those of ribosome minus samples were subtracted, are shown in left panels. Molecular Cell 2012 47, 863-872DOI: (10.1016/j.molcel.2012.06.034) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Elongation Arrest of mifM Generates Polypeptidyl-tRNAs of Different Lengths In vitro translation products of FLAG-mifM (wild-type; lane 1) and its cysteine substitution mutants (lanes 2–7) were analyzed by Nu-PAGE electrophoresis followed by northern hybridization using a DNA probe complementary to tRNACys (upper panel) and by anti-FLAG western blotting (lower panel). Amino acid resides that were subjected to cysteine substitution are indicated by codon numbers at the bottom. “MifM” and “MifM′-tRNA” indicate the translation-completed MifM protein and the stalled nascent chains, respectively. Molecular Cell 2012 47, 863-872DOI: (10.1016/j.molcel.2012.06.034) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Functional Importance of the Negative Charges near the C Terminus of MifM (A) Amino acid sequence of a MifM C-terminal region. Residues previously assigned as important for the elongation arrest are underlined. The consecutive four acidic residues, which are evolutionarily conserved, are highlighted in reverse. (B and C) Effects of substitutions of the acidic residues on the functionality of MifM. Constructs of mifM-yidC2′-lacZ with indicated mifM mutations were introduced into wild-type and ΔspoIIIJ B. subtilis strains. Activities of β-galactosidase (mean ± SD, n = 3) expressed from the yidC2′-lacZ fusion gene in the wild-type (white) and the ΔspoIIIJ (black) backgrounds are shown. The mifM mutations are indicated by amino acids that substituted for the DEED motif of MifM. (D and E) Toeprint analysis of the mifM mutants. Toeprint assays were carried out as described in Figure 1 using the templates shown at the bottom (see above for the notations). (F) Plate tests for the inducibility of yidC2, preceded in cis by mifM having a DEED mutation. β-galactosidase activities in wild-type and ΔspoIIIJ backgrounds were tested by growing the strains bearing mifM-yidC2′-lacZ on X-gal plates. (G) Correlation between the numbers of negative charges at residues 86–89 and the yidC2 inducibility. β-galactosidase activities (mean ± SD, n = 3) are shown for mutants with the indicated DEED alterations. Molecular Cell 2012 47, 863-872DOI: (10.1016/j.molcel.2012.06.034) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Alanine Insertion Analysis of the Integrity of the Mid-Tunnel Arrest Motif (A) Alanine insertions examined. The positions of alanine insertions into the MifM sequence are shown by arrowheads, together with the residues (in numbers) N-terminally next to the insertions. (B) Inducibility of yidC2 in vivo (upper panel) and elongation arrest in vitro (lower panel). Activities of β-galactosidase (mean ± SD, n = 3) expressed from the mifM-yidC2′-lacZ constructs with the indicated mifM insertion mutations in wild-type (white) and the ΔspoIIIJ (black) backgrounds were measured (upper panel). For in vitro translation reactions, FLAG-mifM-myc (wild-type) and its mutants as indicated were used as templates. Samples were treated with RNase A as indicated and analyzed by Nu-PAGE separation and anti-FLAG western blotting (lower panel). “full” and “arrest” indicate the full length and the elongation-arrested products of the MifM derivatives, respectively. Molecular Cell 2012 47, 863-872DOI: (10.1016/j.molcel.2012.06.034) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Nascent MifM Chains Impair the PTC Functions (A) Termination failures within the MifM coding sequence. Toeprint assays were carried out as described in Figure 1, using mifM templates having the UAA termination codon at the indicated positions. (B) Elongation-arresting and termination-arresting functions of MifM correlate with each other. Toeprint assays were carried out for mifM-A90stop, with (lane 2) or without (lane 1) the arrest-impairing second mutation, I70A. (C) Reactivity of nascent MifM′-tRNAs with puromycin. The template FLAG-mifM (lanes 1 and 2) and its derivatives with the mutations indicated at the bottom (lanes 3–8) were translated in vitro at 37°C for 20 min. Subsequently, incubation was continued for an additional 3 min in the presence (even-numbered lanes) or the absence (odd-numbered lanes) of 1 mg/ml puromycin. Reaction products were analyzed by Nu-PAGE electrophoresis and anti-FLAG western blotting (upper panel), as well as by northern blotting using a DNA probe complementary to tRNAAsp (lower panel). “MifM”, “MifM′-tRNA”, and “MifM′-Puro” indicate the full-length protein, elongation-arrested polypeptidyl-tRNA, and its reaction product with puromycin, respectively. Molecular Cell 2012 47, 863-872DOI: (10.1016/j.molcel.2012.06.034) Copyright © 2012 Elsevier Inc. Terms and Conditions