Figure 4.
Fitting of synchronized fluorescence intensity traces of dual-labeled EF-TuAV-33/351·GTP·Phe-tRNAPhe interacting with the 70SIC. (A) The fitting model consists of three EF-Tu-containing ribosome complexes A, B and C, which are preceded by EF-Tu binding to the ribosome and terminated by dissociation. The graphics in A illustrate the essentials of the synchronization of events and averaging of traces with the predicted number of steps of TC binding to the 70SIC. The synchronization analysis was applied to traces of events lasting for 33 ms or more. Pre-synchronized (black) and post-synchronized (blue) Cy3 (C, F) and Cy5 (D, G) intensity symbols were summed up to give the total fluorescence intensity (B, E) squares obtained with cognate (B–D) or near-cognate (E–G) mRNA. All of the intensities are absolute intensities in thousands of camera digitizer units. The blue and black arrows indicate the trigger points and plotting directions. The red lines are the global fits according to the model shown in (A). For the near-cognate data, molecules for which EAppfinal – EAppinitial > 0.05 were grouped and synchronized for this analysis. The calculated values are presented in Table 1 (see also Supplementary Figure S3). Event numbers are as in Figures 2 and 3.