EF-G:A-Site Trna Distance Changes during Translocation

Abstract

Prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs during the polypeptide elongation cycle. However, the kinetic pathway of this important step has not been fully elucidated. Here we describe the application of single molecule fluorescence resonance energy transfer (smFRET) to probe the mechanism of this key process in real time. Attaching a fluorescent probe within domain IV of EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA. Two different sites on L11, position 38 or position 87, as well as two different A-site tRNAs, Phe-tRNAPhe or Val-tRNAVal were labeled with Cy5. The EF-G:L11 FRET efficiency is constant during EF-G occupancy of the ribosome (300 - 400 ms), whereas the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. For both tRNAs, the EF-G:tRNA pair spends most of its time in the intermediate FRET state, with shorter lifetimes in the high and low FRET states. Each transition in the FRET value corresponds to a relative distance change of about 5 to 7 A, so in total, we capture a 12 A relative movement of A-site tRNA away from EF-G during translocation. EF-G:A-site tRNA FRET is sensitive to buffer conditions, with high Mg2+ and polyamines stabilizing the high FRET state. In addition, the translocation inhibitors viomycin and spectinomycin block formation of the low FRET state, although spectinomycin allows more movement between EF-G and tRNA than viomycin. Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.