EMBO Conference on Protein Synthesis and Translational Control

JAMES MUNRO
17
Speaker Abstracts
Single-molecule observations of rate-limiting conformational events during
ribosomal translocation
Kevin Sanbonmatsu 1, Chang-Shung Tung 1, Jamie Cate 2, James Munro 3, Scott Blanchard 3,
Roger Altman 3, Michael Wasserman 3
1 Los Alamos National Laboratory, United States of America
2 UC Berkeley, United States of America
3 Weill Cornell Medical College, United States of America
The mechanism by which tRNA and mRNA translocate with respect to the ribosome is critical
to gene expression. Contemporary models posit a multi-step, kinetically-driven translocation
mechanism, rate-limited by conformational processes in the ribosome and elongation factor G
(EF-G). However, the precise order and timing and specific roles of distinct conformational
degrees of freedom in the ribosome complex remain open questions that have thus far been
difficult or impossible to delineate. Here, using single-molecule fluorescence resonance energy
transfer (smFRET), we have investigated the order and timing of conformational changes in the
ribosome complex critical to tRNA-mRNA movements. By obtaining data from multiple
structural perspectives we have directly monitored the events leading up to formation of the
unlocked ribosome conformation, in which the P/E hybrid state is formed, the small ribosomal
subunit is ratcheted, and the L1 stalk has adopted a closed conformation. These results reveal
that conformational processes on path to the unlocked state do not occur in a concerted
fashion, but are instead loosely coupled. Under equilibrium conditions, tRNAs exchange
between hybrid and classical states rapidly, subunit ratcheting-unratcheting exchange occurs
relatively slowly, and unlocked state formation is rate-limited by L1 stalk closure. The loosely
coupled nature of these events specifies that multiple structural pathways may be transited
during translocation. Direct observations of EF-G binding and translocation events suggest that
EF-G captures the unlocked state of the ribosome, inhibiting its return to the locked
configuration and promoting a fast dynamic mode of the L1 stalk. In this model, opening of the
L1 stalk on the ratcheted/P/E hybrid state ribosome contributes directly to movement of P-site
tRNA to the E site.