Single-molecule experiments using TIRF microscopy
This protocol is extracted from research article:
EF-G–induced ribosome sliding along the noncoding mRNA
Sci Adv, Jun 5, 2019; DOI: 10.1126/sciadv.aaw9049

Single-molecule FRET experiments were carried out at 22°C or 4° to 10°C temperature as stated. The stalled ribosome complexes were diluted in HiFi buffer to a final concentration of 1 nM and immobilized on biotin–polyethylene glycol quartz slides preincubated with NeutrAvidin (Thermo Fisher Scientific) using the mRNA annealed to a biotinylated primer. The imaging buffer was HiFi containing 3.5 mM MgCl2 supplemented with an oxygen-scavenging system (5 mM protocatechuic acid and 50 nM protocatechuate-3,4-dioxygenase from Pseudomonas) and a triplet-state quencher mixture (1 mM Trolox and 1 mM methylviologen) (Sigma-Aldrich), as described (37). smFRET experiments were performed on an IX81 inverted objective-based TIRF microscope with a 100× 1.45 numerical aperture oil immersion objective (PLAPON, Olympus). A charge-coupled device C9100-13 camera (Hamamatsu) was used for recording images at a time resolution of 30 frames/s. To image complexes at low temperature, we used an aluminum alloy cube that was cooled on ice and placed on the microscope slide during the measurements. The temperature was controlled and maintained constant within approximately 1°C. Fluorescence time traces for donor (Cy3) and acceptor (Cy5) were extracted and analyzed using custom-made MATLAB (MathWorks) software according to published protocols (37). The distribution of FRET states shown in the state histograms was fitted to a sum of Gaussian functions using a nonlinear minimization procedure (fminsearch, MATLAB). The R2 value for all fits was larger than 0.98. FRET states and corresponding population values were defined from three independent datasets and presented as means ± SD in table S1. For normalization shown in Fig. 4F, we estimated that about 37% of complexes can change the conformation from the nonrotated to either rotated or hyper-rotated under the conditions of the smFRET experiment (Fig. 4B), consistent with the bypassing efficiency (40%) upon temperature shift (Fig. 1C), whereas the remaining fraction remains in the nonrotated state and was omitted from the calculations.

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