Figure 4.

NAc-Phe-tRNA^Phe titration in the 3’ pyrene-labelled mRNA+10 based translocation reaction in stopped-flow

(A) Pyrene fluorescence traces obtained by adding NAc-Phe-tRNA^Phe (0.5–5 µM) in the elongation mix (EM), which was mixed rapidly with the 70S IC (0.5 µM) carrying mRNA+10 and tRNA^fMet. (B) The plot of the observed rates of pyrene fluorescence change in (A) versus NAc-Phe-tRNA^Phe concentration. The data are fitted with linear equation to obtain the binding parameters. The K_D was determined as k_off/k_on = 2.7 ± 0.2 µM. (C) Pyrene fluorescence traces obtained by adding NAc-Phe-tRNA^Phe (1–5 µM) in the initiation mix (IM) containing 70S IC (0.5 µM), which was mixed rapidly with EM containing EF-G (5 µM). The start point of each fluorescence trace is offset vertically for easy comparison. The data is fitted with double exponential function and presented in Supplementary Table 3. (D) Correlation of the calculated and experimentally determined fractions of the 70S pre-TC, obtained from the K_D value (B) and fluorescence amplitudes (C), respectively. Data represent average of three replicates with standard deviation.