(A,B) In the presence of GDPNP. (C,D) In the presence of GTPγS. (E,F) With RF3(H92A) and GTP. (A,C,E) Dissociation of RF1 monitored by FRET between RF1-Cy5 (10 nM) and L11-Cy3 in the presence of excess RF3 (1 µM). The nucleotide concentration is 1 mM. Time courses were synchronized to the onset of the FRET event and combined in contour plots. FRET values (mean ±sd, from three independent data sets) are 0.47 ± 0.04 and 0.67 ± 0.04 (A), 0.66 ± 0.04 (C), and 0.51 ± 0.03 (E). (B,D,F) Dwell time distributions and the rates of RF1 and RF3 dissociation and subunit rotation.