Figure 5. Single-molecule time traces of S6-Cy5/L9-Cy3 ribosome complexes showing intersubunit movement induced by (a) reaction with puromycin, (b) EF-G binding or (c) translocation.

(a) Ribosomes containing fMet-tRNA^fMet bound to the P site were reacted with puromycin (1 mM) in buffer B (see Experimental procedures), flowed into the microscope slide ~20 s after beginning the fluorescence recording (arrow). Intersubunit movement is seen as a sharp decrease in the FRET signal (indicated by the vertical dashed line). 13 of 24 molecules that did not photobleach before addition of puromycin showed similar behavior with an average deacylation time of ~20s. (b) EFG-GDPNP (300 nM, 250 µM) in buffer B (see Experimental procedures) was introduced at ~40s (arrow) into complexes containing deacylated tRNA^fMet bound to the P site and a vacant A site. EF-G-induced intersubunit rotation is observed as stabilization of the low FRET state (vertical dashed line). 42 of 51 molecules showed similar behavior with an average time between addition of EFG·GDPNP and the last FRET fluctuation of ~15s (c) EF-G·GTP (300 nM, 250 µM) in buffer B (see Experimental procedures) was introduced at ~20 s (arrow) to pre-translocation complexes containing deacylated tRNA^fMet bound to the P site and N-Ac-Phe-tRNA^Phe bound to the A site. Translocation is observed as the transition to the high FRET state (vertical dashed line). 40 of 76 molecules showed similar behavior with an average translocation time of ~22s after addition of EF-G·GTP. For further information regarding the flow experiments, see Experimental Procedures.