Fig. 1: Toeprinting signal of 30S complexes assembled on various mRNAs.
aToeprint signals for (1) 30S:mRNA, (2) 30S:mRNA:Met-tRNAiMet, (3) 30S:mRNA:aIF2:Met-tRNAiMet (4) 30S:mRNA:aIF2:Met-tRNAiMet:aIF1A were measured as described in Methods. Data are presented as means ± SD from independent experimental units (Methods). Dots show individual data points. Typical experiments and the sequences of the mRNAs are shown in Supplementary fig. 3 and Supplementary Table 1. Ss-MAP mRNA has a 5’OH extremity whereas the Ss-aIF2β mRNA has a 5’ triphosphate group. Experimental toeprinting conditions were the same for model-SD mRNA (n = 4), Ss-EF1A-like mRNA (n = 3) and Ss-aIF2β lmRNA (n = 3). With Ss-MAP lmRNA (n = 4), the molar excesses of IF and tRNA with respect to 30S were increased by factors of 4 and 2.5, respectively. b Importance of the 5’-triphosphate end. Relative toeprinting signals were measured for 5’-triphosphate (Ss-aIF2β only), 5’ monophosphate or 5’-OH versions of the Ss-aIF2β and Ss-MAP lmRNAs. The values represented are the means and standard deviations from 3 independent experiments. For each mRNA, values were normalized to the mean obtained with the monophosphorylated mRNA which was given the arbitrary value of 100%. Two-tailed P values were calculated from unpaired t tests in PRISM (P values were 0.0014 and 0.0038 for Ss-aIF2β and 0.017 for Ss-MAP). Typical experiments are shown in Supplementary fig. 17. c Influence of Ss-eS26 concentration on the main toeprinting signal intensity obtained with 30S:mRNA:aIF2:Met-tRNAiMet complexes. Ss-eS26 concentrations ranged from 0 to 1.2 µM. 30S concentration in all experiments was 0.1 µM (Supplementary fig. 20). The means from independent experiments (left: n = 4; middle: n = 2; right: n = 3) with the calculated standard deviations are represented. Dots show individual data points. Source data are provided as a Source Data file.
