Fig. 1: Experimental design for single-molecule tracking.
From: A complex between IF2 and NusA suggests early coupling of transcription-translation
a Schematic of the main steps and key intermediates in canonical translation initiation. b Two approaches for fluorescence labeling were used: (upper) IFs fused to the HaloTag protein were expressed at low concentration from a plasmid with subsequent coupling to the JFX549 fluorophore in vivo, and (lower) labeling of fMet-tRNAfMet in vitro with the Cy5 fluorophore, followed by the delivery of labeled molecules via electroporation. c HMM fitting of single-molecule trajectories of HaloTag-IF3 using models with 1–9 states (Supplementary Data 3). Each model is arranged along the y axis, with discrete diffusion states represented by circles positioned along the x-axis according to their diffusion coefficient. The area of each circle represents the relative occupancy of different diffusional states. Red dashed lines indicate thresholds at 1 µm²/s and 8 µm²/s, used to distinguish ribosome-bound states (cyan area) from free factors (magenta area) and cleavage products. d Phase contrast images and fluorescence time-lapse movies were acquired for mini-colonies of cells that were recovered after labeling. These images were used for cell segmentation and for constructing single-molecule diffusion trajectories within the corresponding cells. Cell outlines are shown in orange. Beginning and the end of the trajectories are shown with red and blue circles, respectively. The figure shows a diffusion trajectory of HaloTag-IF3 (see also Movie S4), color coded with respect to binding state (cyan and magenta). Source data are provided as a Source Data file.
