Volume 68, Issue 3, Pages 528-539.e5 (November 2017) Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing  Yuhei Chadani, Tatsuya Niwa, Takashi Izumi, Nobuyuki Sugata, Asuteka Nagao, Tsutomu Suzuki, Shinobu Chiba, Koreaki Ito, Hideki Taguchi  Molecular Cell  Volume 68, Issue 3, Pages 528-539.e5 (November 2017) DOI: 10.1016/j.molcel.2017.10.020 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 528-539.e5DOI: (10.1016/j.molcel.2017.10.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Translation of Negative Charge Clusters Destabilizes the 70S Ribosome (A) Schematic representation of Maa-10X. Homo-decamers of amino acids (10X) were inserted between Maa and LacZα. (B) Polypeptidyl-tRNA accumulation by 10E, 10D, and 10P. Maa-10X constructs were translated using PUREfrex and 35S-methionine, treated with puromycin (Pm) as indicated, and separated by neutral pH SDS-PAGE with optional RNase A (RN) pretreatment. Radioactive full-length proteins are indicated by “CC” (completed chain). Polypeptidyl-tRNAs and tRNA-cleaved polypeptides are indicated by schematic labels in orange. (C) Pth sensitivity of polypeptidyl-tRNAs. Translation in PUREfrex or S30, as indicated, was followed by optional incubation with purified EF-P (EFP), puromycin (Pm), or purified peptidyl-tRNA hydrolase (Pth). Samples in SDS were treated with RNase A (RN), as indicated. (D) Translation anomaly dependence on the release and recycling factors. Maa-10E was translated in the presence (+) or the absence (–) of release (RF1, -2, and -3) and recycling (RRF) factors as indicated on the top. Asterisk indicates the termination-arrested polypeptidyl-tRNAs. (E and F) Sedimentation behaviors of the translation products in the reaction mixtures. GFP-10E (E) and SecM (F) were translated using Cy5-Met-tRNA for initiation, fractionated by sucrose gradient centrifugation, separated by SDS-PAGE, and detected by fluorescence. Distributions of the bulk ribosomes were determined by A254 measurements (see Figure S1E). See also Figure S1. Molecular Cell 2017 68, 528-539.e5DOI: (10.1016/j.molcel.2017.10.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Ribosome Destabilization Sequences (A) Effects of N-terminally adjacent homo-decamers on the negative-charge-dependent translation abortion. Extents of translation abortion (Pth sensitivity index; see STAR Methods) are shown for Maa-10X-10E, whose sequences are shown at the bottom. Note that 10R-10E was evaluated by the RNase sensitivity because it exhibited significant elongation arrest. (B) Sedimentation of the GFP-10K-10E translation products. The GFP-10K-10E translation was followed by incubation with Pth for the bottom sample. Translation products were detected by anti-His6 western blotting. (C) Translation abortion by 5(DP). Maa-5(DX) translation was followed by optional incubation with Pth. (D) PI values of Maa-5(DX) proteins. (E) Effects of EF-P on Maa-5(DP) translation. Values were obtained from three independent experiments. See also Figure S2. Molecular Cell 2017 68, 528-539.e5DOI: (10.1016/j.molcel.2017.10.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 In Vivo IRD and a Counteracting Role of bL31 (A) YagN translation. In vitro translation of His6-YagN in the presence of 35S-methionine was followed by optional incubation with puromycin (Pm: lane 2) or Pth (lane 3). Products were electrophoresed after optional RNase A treatment (RN). In parallel, His6-YagN was induced briefly in vivo, pulse-labeled with 35S-methionine, and affinity isolated (lanes 5 and 6; lane 6, RNase A treated). Radioactive materials were detected after neutral pH SDS-PAGE. Polypeptidyl-tRNA, its peptide moiety, methionyl-tRNA, and in vivo backgrounds (including SlyD) are indicated by the orange rectangle with and without schematic tRNA, Met, and asterisks, respectively (Chadani et al., 2016). (B) YagN translation-aborting sequence. Residues 82 to 119 of YagN (shown below the graph) were inserted into Maa-LacZα. Subsequently, frameshift (FS: resulting amino acid sequence is shown at the bottom) and other mutations were introduced. PI values were calculated after in vitro translation of these Maa-YagN constructs. (C) Ribosome occupancy in yagN mRNA. The published ribosome profiling data (Li et al., 2012) are graphically depicted, with an enlargement showing the ribosome-empty region together with the A-site amino acids. (D–F) Translation in vitro and in vivo of rpoD (D), GFP-10E (E), and GFP-10K-10E (F), as carried out in (A). (G) In vivo production of a Pth-sensitive product. GFP-10K-10E was expressed in TS101 (pth(Ts)) cells at 30°C, and pulse-chase labeling was done at this temperature (open circles) or after shift to 42°C for 1 min (solid circles) to inactivate Pth (Cruz-Vera et al., 2000). In each case, pulse labeling for 1 min was followed by chase with unlabeled methionine for the indicated times. His6-tagged proteins were analyzed as in (A), and relative intensities of radioactive polypeptidyl-tRNA are shown. Values over 100% (0 time) may have been due to the residual incorporation of the label. Results of three independent experiments are shown with standard deviation. (H) Enhanced IRD by bL31 deletion. His6-GFP-10E was expressed in cells with indicated deletion in the ribosomal protein gene, pulse labeled, and analyzed as above. (I) Destabilization of the 70S ribosome by the lack of bL31. Exponentially growing cells of wild-type (MG1655) E. coli and the bL31 deletion mutant (CY619; ΔrpmE) were lysed and centrifuged through sucrose gradients. Distributions of the ribosomes were monitored by A254 measurement. See also Figure S3. Molecular Cell 2017 68, 528-539.e5DOI: (10.1016/j.molcel.2017.10.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 MgtL Nascent Chain-Ribosome Complexes as a Mg2+ Sensor for Regulation of MgtA (A) The absence of bL31 leads to a striking overproduction MgtA. We addressed proteomic consequences of the absence of bL31 in E. coli using mass spectroscopy-based quantitative shotgun approach. Mutant/wild-type abundance ratios were determined for 584 proteins, which are aligned graphically. Increases and decreases of more than 2-fold (see Table S1) are shown in red and blue, respectively. (B) The mgtL-mgtA operon and the amino acid sequence of MgtL. One of the alternative mRNA secondary structures in the intergenic region (Park et al., 2010) is shown schematically. (C) Top: MgtL translation abortion in vitro and amino acid residues responsible for it. MgtL and its indicated mutants were translated in PUREfrex in the presence of 35S-methionine followed by optional incubation with Pth. Samples in SDS were treated with RNase A, as indicated, before electrophoresis. The size of aborted polypeptides (after removal of tRNA) were too small for detection by the gel system. Note that the lower band represents Met-tRNA. Bottom left: factor dependence of the MgtL translation abortion. MgtL was translated in PUREfrex with and without RF1, RF2, RF3, and RRF (ΔRFs). Bottom right: EF-P effects on MgtL translation. MgtL was translated in PUREfrex with optional inclusion of EF-P. (D) MgtL translation abortion in vivo. MgtL-LacZ, with indicated mutations in mgtL, was expressed in E. coli and β-galactosidase activities were determined. m.u., Miller unit (Miller, 1972). (E) Effects of Mg2+ on in vitro translation. MgtL (left), Maa-10E (middle), and SecM (right) were translated in vitro using PUREfrex supplemented with 35S-methionine and additional concentrations of Mg2+, as indicated. Increasing the Mg2+ concentration by +8 mM reduced the overall translation by ∼55%. Proportions of peptidyl-tRNA in the translation products are shown; note that MgtL contains only single methionine at its start. (F) N-terminal MgtL sequences required for the Mg2+-responsive translation abortion. Indicated numbers of N-terminal residues (FL, full-length) of MgtL were fused to the N terminus of LacZ. Resulting fusion proteins were expressed in E. coli growing on M9 medium supplemented with 10 mM (high) or 10 μM (low) Mg2+ for measurement of β-galactosidase activities (left). Mg2+ effects are reported in the right panel as the LacZ activity ratios (low Mg2+/high Mg2+). (G) N-terminal MgtL sequences required for the Mg2+-responsive regulation of MgtA expression. The mgtL-mgtA′-lacZ reporter, in which LacZ was fused in frame to the N-terminal region of MgtA, and its derivatives with the indicated mutation in mgtL were expressed in E. coli growing in medium containing 10 mM or 10 μM Mg2+ for measurement of β-galactosidase activities (left). LacZ activities of the reporters with indicated mutations in mgtL were measured in E. coli growing in the presence of 50 μM Mg2+, which proved sufficient to give the basal (repressed) level expression of MgtA (right). See also Figure S4. Molecular Cell 2017 68, 528-539.e5DOI: (10.1016/j.molcel.2017.10.020) Copyright © 2017 Elsevier Inc. Terms and Conditions