Fig. 1: EF-G-induced translocation of pept-tRNA.
a Schematic of the smFRET experiment. Pept-tRNALys-Cy5 (red star) fluctuates between A/A, A/P, and A/P* states in the absence of EF-G. Upon EF-G addition, pept-tRNA moves into a transient CHI state before reaching the P/P state. The movement is monitored by change in FRET between pept-tRNALys-Cy5 and L11-Cy3 (green star). FRET values are assigned to PRE and POST states in independent experiments (Supplementary Fig. 1) and validated by distance measurements based on cryo-EM structures19,24,25. b Coding sequences of mRNA constructs. Slippery sequence encodes fMet-Gly-Lys-Phe peptide in 0 frame and fMet-Gly-Lys-Val in −1 frame. Non-slippery mRNA that does not support frameshifting encodes only the 0-frame peptide fMet-Phe-Lys-Phe. c Representative smFRET time trace showing tRNA translocation on non-slippery mRNA. Trace shows fluctuations between FRET 0.8 (A/A, A/P) and 0.6 (A/P*) followed by rapid transition to FRET 0.2 (P/P). Black line shows the HMM fit of the data here and in all smFRET traces. d Contour plot showing distribution of FRET values during tRNA translocation on non-slippery mRNA. Transitions occur from either A/A and A/P or A/P* to P/P in less than 33 ms. Traces are synchronized to the first transition below FRET 0.5. Histogram at the right shows distribution of FRET values after synchronization. Data are from 5 independent experiments (N = 5). e Representative smFRET time trace showing tRNA translocation on slippery mRNA. Trace shows rapid transition from FRET 0.8 (A/A, A/P) to FRET 0.4 (CHI)23,46, followed by fluctuations between FRET 0.4 and 0.2 before adopting a long-lived FRET 0.2 state (P/P). f Contour plot showing distribution of FRET values during translocation on slippery mRNA. The contour plot contains mixture of trajectories. 81% of traces show direct transitions from A/A, A/P or A/P* to P/P. 19% of traces show transitions from A/A, A/P, or A/P* to CHI followed by fluctuations between CHI and P/P states before transition to long-lived P/P state. Traces are synchronized to the first transition below FRET 0.5. Histogram at the right shows distribution of FRET values after synchronization. Data are from 12 independent experiments (N = 12).
