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Fluctuations of Transfer RNAs between Classical and Hybrid States

Harold D. Kim ^*, Joseph D. Puglisi  and Steven Chu ^*  

^* Department of Physics and Department of Structural Biology, Stanford University, Stanford, California 94305; and Lawrence Berkeley National Laboratory and Departments of Physics and Molecular and Cellular Biology, University of California, Berkeley, California 94720

Correspondence: Address reprint requests to Steven Chu, E-mail: schu{at}lbl.gov.

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.

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