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Mutations of MifM residues 41–60 compromise elongation arrest in vitro . In vitro translation using Bs hybrid PURE system was directed by the gfp–mifM 35–95 –myc templates with or without the mifM mutations affecting residues 41–60, indicated at the bottom. Reaction products were analyzed by <t>SDS-PAGE</t> using the neutral pH (WIDE-RANGE) gel system, followed by immunoblotting with anti-GFP (upper panel) and anti-Myc (lower panel). To remove the tRNA moiety from polypeptidyl-tRNA, portions of samples were treated with RNaseA before electrophoresis (lanes with even-numbers). Black and white triangles indicate full-length (GFP-MifM-Myc) and arrested (GFP-MifM’) forms of the translation product, respectively. Full-length blots are presented in Supplementary Fig. 2B .
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1) Product Images from "MifM-instructed translation arrest involves nascent chain interactions with the exterior as well as the interior of the ribosome"

Article Title: MifM-instructed translation arrest involves nascent chain interactions with the exterior as well as the interior of the ribosome

Journal: Scientific Reports

doi: 10.1038/s41598-018-28628-y

Mutations of MifM residues 41–60 compromise elongation arrest in vitro . In vitro translation using Bs hybrid PURE system was directed by the gfp–mifM 35–95 –myc templates with or without the mifM mutations affecting residues 41–60, indicated at the bottom. Reaction products were analyzed by SDS-PAGE using the neutral pH (WIDE-RANGE) gel system, followed by immunoblotting with anti-GFP (upper panel) and anti-Myc (lower panel). To remove the tRNA moiety from polypeptidyl-tRNA, portions of samples were treated with RNaseA before electrophoresis (lanes with even-numbers). Black and white triangles indicate full-length (GFP-MifM-Myc) and arrested (GFP-MifM’) forms of the translation product, respectively. Full-length blots are presented in Supplementary Fig. 2B .
Figure Legend Snippet: Mutations of MifM residues 41–60 compromise elongation arrest in vitro . In vitro translation using Bs hybrid PURE system was directed by the gfp–mifM 35–95 –myc templates with or without the mifM mutations affecting residues 41–60, indicated at the bottom. Reaction products were analyzed by SDS-PAGE using the neutral pH (WIDE-RANGE) gel system, followed by immunoblotting with anti-GFP (upper panel) and anti-Myc (lower panel). To remove the tRNA moiety from polypeptidyl-tRNA, portions of samples were treated with RNaseA before electrophoresis (lanes with even-numbers). Black and white triangles indicate full-length (GFP-MifM-Myc) and arrested (GFP-MifM’) forms of the translation product, respectively. Full-length blots are presented in Supplementary Fig. 2B .

Techniques Used: In Vitro, SDS Page, Electrophoresis

Electrophoretic visualization of the in vivo translation products. The gfp-mifM-lacZ gene fusion (lanes 1 and 4) and its mutant forms, fs41–60 (lanes 2 and 5) and GS41–60 (lanes 3 and 6), were expressed in B. subtilis , and the arrested (GFP-MifM’) and the full-length (GFP-MifM-LacZ) translation products were detected by anti-GFP (lanes 1–3) and anti-LacZ (lanes 4–6) immunoblotting, respectively, after separation by SDS-PAGE. The tRNA moiety of the arrest product had been removed under the sample preparation conditions used. Full-length blots are presented in Supplementary Fig. 2A .
Figure Legend Snippet: Electrophoretic visualization of the in vivo translation products. The gfp-mifM-lacZ gene fusion (lanes 1 and 4) and its mutant forms, fs41–60 (lanes 2 and 5) and GS41–60 (lanes 3 and 6), were expressed in B. subtilis , and the arrested (GFP-MifM’) and the full-length (GFP-MifM-LacZ) translation products were detected by anti-GFP (lanes 1–3) and anti-LacZ (lanes 4–6) immunoblotting, respectively, after separation by SDS-PAGE. The tRNA moiety of the arrest product had been removed under the sample preparation conditions used. Full-length blots are presented in Supplementary Fig. 2A .

Techniques Used: In Vivo, Mutagenesis, SDS Page, Sample Prep

The absence of the MifM region N-terminal to residue 60 compromises the elongation arrest in vivo and in vitro . ( A ) Arrest efficiencies of MifM mutants lacking N-terminal sequences. β-galactosidase activities (mean ± s.d., n = 3) of cells harboring Pgrac-mifM-lacZ derivatives with the N-terminal truncations of MifM, with or without additional arrest-impairing mutation (AAAA) at the PTC-proximal region, were measured. The ratio of β-galactosidase activity (WT/AAAA) is shown for each N-terminal mutation. The numbers below the graph indicate the first MifM residues in the truncated MifM mutant derivatives (numbering by the wild-type MifM protein) ( B ) In vitro translation of MifM mutants lacking N-terminal sequences. Similar to ( A ), each N-terminal mutation was combined with a mutation I70A at a mid-tunnel region, which impairs the arrest partially (this mutation was used as it did not affect the electrophoretic mobility of MifM). DNA templates, with the N-terminal truncations indicated by the residues that now became the first mifM -encoded residues, were used to direct in vitro translation with Bs hybrid PURE system in the presence of 35 S-methionine. Samples were treated with RNase A, separated by SDS-PAGE and radioactive products were analyzed with a phosphorimager. Black and white triangles indicate full-length (MifM-Myc) and arrested (MifM’) forms of the translation products, respectively. Full-length gel is presented in Supplementary Fig. 2C .
Figure Legend Snippet: The absence of the MifM region N-terminal to residue 60 compromises the elongation arrest in vivo and in vitro . ( A ) Arrest efficiencies of MifM mutants lacking N-terminal sequences. β-galactosidase activities (mean ± s.d., n = 3) of cells harboring Pgrac-mifM-lacZ derivatives with the N-terminal truncations of MifM, with or without additional arrest-impairing mutation (AAAA) at the PTC-proximal region, were measured. The ratio of β-galactosidase activity (WT/AAAA) is shown for each N-terminal mutation. The numbers below the graph indicate the first MifM residues in the truncated MifM mutant derivatives (numbering by the wild-type MifM protein) ( B ) In vitro translation of MifM mutants lacking N-terminal sequences. Similar to ( A ), each N-terminal mutation was combined with a mutation I70A at a mid-tunnel region, which impairs the arrest partially (this mutation was used as it did not affect the electrophoretic mobility of MifM). DNA templates, with the N-terminal truncations indicated by the residues that now became the first mifM -encoded residues, were used to direct in vitro translation with Bs hybrid PURE system in the presence of 35 S-methionine. Samples were treated with RNase A, separated by SDS-PAGE and radioactive products were analyzed with a phosphorimager. Black and white triangles indicate full-length (MifM-Myc) and arrested (MifM’) forms of the translation products, respectively. Full-length gel is presented in Supplementary Fig. 2C .

Techniques Used: In Vivo, In Vitro, Mutagenesis, Activity Assay, SDS Page

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