Figure 6.

EttA-mediated regulation of the open L1 stalk ↔ closed L1 stalk equilibrium of a PRE^-A_fMet complex as observed using smFRET_L1-L9. (a) Cartoon diagram of the conformational equilibrium of the PRE^-A_fMet complex between the MS-I conformation harboring an open L1 stalk (ribosomal complex on left) and the MS-II conformation harboring a closed L1 stalk (ribosomal complex on right). 30S subunit, tan cartoon; 50S subunit, blue cartoon; tRNA^fMet, green ribbon,; mRNA, black curve; Cy3 FRET donor fluorophore, green circle; and Cy5 FRET acceptor fluorophore, red circle. (b, c, d) smFRET_L1-L9 experiments recorded in the presence of 2 mM ATP and in (b) the absence of EttA, (c) the presence of 6 µM EttA, or (d) the presence of 6 µM EttA-EQ₂. 1^st row: Representative Cy3 and Cy5 fluorescence intensity (Fluor. int.) vs. time trajectories. The fluorescence intensities are plotted in arbitrary units (a.u.) with the Cy3 fluorescence intensity plotted in green and the Cy5 fluorescence intensity plotted in red. 2^nd row: The corresponding E_FRET vs. time trajectories. The E_FRET at each time point was calculated using E_FRET = I_Cy5 / (I_Cy3 + I_Cy5), where I_Cy3 and I_Cy5 are emission intensities of Cy3 and Cy5, respectively, and is plotted in blue. 3^rd row: Surface contour plots of the time evolution of the population FRET. The contour plots were generated by superimposing individual E_FRET vs. time trajectories, and colored from white (lowest-populated) to red (highest-populated). n denotes the number of E_FRET vs. time trajectories used to construct each contour plot.