Fluctuations of Transfer RNAs between Classical and Hybrid States

Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic equilibrium between two different conformations called classical and hybrid states before translocation. Here, we have used single-molecule fluorescence resonance energy transfer to study the effect of Mg2+ on tRNA dynamics with and without an acetyl group on the A-site tRNA. When the A-site tRNA is not acetylated, tRNA dynamics do not depend on [Mg2+], indicating that the relative positions of the substrates for peptide-bond formation are not affected by Mg2+. In sharp contrast, when the A-site tRNA is acetylated, Mg2+ lengthens the lifetime of the classical state but does not change the lifetime of the hybrid state. Based on these findings, the classical state resembles a state with direct stabilization of tertiary structure by Mg2+ ions whereas the hybrid state resembles a state with little Mg2+-assisted stabilization. The antibiotic viomycin, a translocation inhibitor, suppresses tRNA dynamics, suggesting that the enhanced fluctuations of tRNAs after peptide-bond formation drive spontaneous attempts at translocation by the ribosome.