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dc:title: Figure S1
modified: 2014-08-28T09:28:20
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dc:description: Overall elongation rate as measured for a model protein in vitro. Kinetics of CspA translation in vitro at different temperatures. CspA mRNA, which codes for a 70 aa-long protein from E. coli, was prepared by T7 RNA-polymerase transcription. Ribosomes were synchronized by forming an initiation complex consisting of 70S ribosomes, CspA mRNA and a fluorescence derivative of initiator tRNAfMet carrying BodipyFL at the ?-amino group of Met in the presence of initiation factors (IF1, IF2, and IF3) and GTP. Translation was carried out in a fully reconstituted translation system by adding initiation complexes (15 nM) to a mixture of EF-Tu?GTP?aminoacyl-tRNA (40 然 aminoacyl-tRNA, 100 然 EF-Tu in total), EF-G (3 然), GTP (2 mM), phosphoenol pyruvate (6 mM), and pyruvate kinase (0.1 mg/ml) in HiFi buffer (50 mM Tris-HCl, pH 7.5, 30 mM KCl, 70 mM NH4Cl, 3.5 mM free MgCl2, 0.5 mM spermidine, and 8 mM putrescine) at the indicated temperatures [46]. In the absence of translation termination and ribosome recycling factors, translation was limited to a single round, i.e. at most one molecule of CspA was synthesized per ribosome. The reactions were stopped at the indicated time intervals and translation products separated on 16.5% Tris-Tricine-PAGE and visualized by the fluorescent reporter BODIPY-Fl at the N-terminus of the peptides [47] (left panels). The intensity of the full length product was quantified with ImageJ (right panel, circles). Average translation rates per codon, which depend on the elongation rates only, were determined by exponential fitting (fits in graphs of the right panel).(TIF)
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description: Overall elongation rate as measured for a model protein in vitro. Kinetics of CspA translation in vitro at different temperatures. CspA mRNA, which codes for a 70 aa-long protein from E. coli, was prepared by T7 RNA-polymerase transcription. Ribosomes were synchronized by forming an initiation complex consisting of 70S ribosomes, CspA mRNA and a fluorescence derivative of initiator tRNAfMet carrying BodipyFL at the ?-amino group of Met in the presence of initiation factors (IF1, IF2, and IF3) and GTP. Translation was carried out in a fully reconstituted translation system by adding initiation complexes (15 nM) to a mixture of EF-Tu?GTP?aminoacyl-tRNA (40 然 aminoacyl-tRNA, 100 然 EF-Tu in total), EF-G (3 然), GTP (2 mM), phosphoenol pyruvate (6 mM), and pyruvate kinase (0.1 mg/ml) in HiFi buffer (50 mM Tris-HCl, pH 7.5, 30 mM KCl, 70 mM NH4Cl, 3.5 mM free MgCl2, 0.5 mM spermidine, and 8 mM putrescine) at the indicated temperatures [46]. In the absence of translation termination and ribosome recycling factors, translation was limited to a single round, i.e. at most one molecule of CspA was synthesized per ribosome. The reactions were stopped at the indicated time intervals and translation products separated on 16.5% Tris-Tricine-PAGE and visualized by the fluorescent reporter BODIPY-Fl at the N-terminus of the peptides [47] (left panels). The intensity of the full length product was quantified with ImageJ (right panel, circles). Average translation rates per codon, which depend on the elongation rates only, were determined by exponential fitting (fits in graphs of the right panel).(TIF)
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dcterms:created: 2014-08-28T09:28:20
dcterms:modified: 2014-08-28T09:28:20
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title: Figure S1
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