Fig. 3. An RNC-SRP binding assay shows that NAC co-binds with SRP on the ribosome.

a Scheme of the FRET assay to measure RNC-SRP binding. RNC was labeled with BDP (cyan star) at the N-terminus of signal sequence, and SRP was labeled with TMR (green star) at residue 12 of SRP54. b Fluorescence emission spectra showing FRET between RNC(ssmt)^BDP and SRP^TMR, using an excitation wavelength of 485 nm. Where indicated, the reactions contained 1 nM RNC(ssmt)^BDP, 5 nM SRP^TMR, and 50 nM unlabeled SRP. c Equilibrium titrations to measure the K_d of the RNC-SRP complex. Titrations contained 1 nM BDP-labeled RNC(ss) or RNC(ssmt) and indicated concentrations of SRP^TMR. FRET efficiency was calculated using the fluorescence emission intensity at 517 nm according to Eq. (2). The lines are fits of the data to Eq. (4), which gave the indicated K_d values. d Effects of NAC and NACmt on the FRET efficiency between RNC^BDP and SRP^TMR. The reactions contained 1 nM BDP-labeled RNC(ss) or RNC(ssmt), 20 nM SRP^TMR, and indicated concentrations of NAC or NACmt. e, f RNC-SRP FRET titrations in the presence of increasing NAC concentrations for RNC(ss) (e) and RNC(ssmt) (f). The titrations contained 1 nM RNC^BDP, indicated concentrations of SRP^TMR, 0–400 nM NAC (e) or 0–62.5 nM NAC (f). The lines are global fits of the data to the model in g using Eq. (8) in the Methods section. g Left panel: model for the coupled binding of SRP and NAC to the RNC. The same coupling factor α describes the degree to which SRP affects the RNC binding affinity of NAC and vice versa, as dictated by the principle of thermodynamic coupling. Right panel: summary of the parameters obtained from global fits of the data in e and f to Eq. (8). All values are reported as optimized value ± square root of covariance (equivalent to fitting error). Source data for b–f are provided in the Source Data file.