Fig. 3: Dependence of mRNA translocation relative to the 30S body on GTP hydrolysis and Pi release.
From: The ribosome derives the energy to translocate and unwind mRNA from EF-G binding
a–c Schematics of the experimental setup (a) and readout under unperturbed (b) and perturbed GTPase conditions (c) for the hairpin unwinding fleezers assay, shown for comparison. d–f Schematics of the experimental setup (d) and readout under unperturbed (e) and perturbed GTPase conditions (f) for the assisting force fleezers assay for simultaneous detection of mRNA translocation relative to the 30S body and EF-G binding during ribosomal translocation. g A fleezers trajectory of consecutive steps taken by a single ribosome in the presence of wild-type EF-G and GTP. The shaded area is magnified in the right panels in which the detected transitions are demarcated by red lines. h Summary of measurements from the hairpin unwinding assay (green bars) and the assisting force assay (cyan bars), represented as mean ± standard error. Individual data points are shown as small circles. The τpre and τunwinding measurements are shown in the left panel, and τpost and τrelease measurements are shown in the right panel for various experimental conditions. The conditions are listed on the left and the number of data points for each condition (N) are shown on the right. Note that the time axis is shown in logarithmic scale to better separate short (normal) and long times, as demarcated by the dashed red lines. For GDPNP and GTPγS, a higher analog:GTP ratio was used in the assisting force assay (2:1) compared to the hairpin unwinding assay (1:2). Fusidic acid (FA) measurements in the hairpin assay (*) were made previously32. Source data are provided as a Source Data file.
