Coupling Of Ribosome And tRNA Dynamics During Translation

Abstract

Comparisons of X-ray crystallographic and cryogenic electron microscopic structures of ribosomal complexes have led to the hypothesis that conformational dynamics of the ribosome, its transfer RNA (tRNA) substrates, and associated translation factors play important mechanistic and regulatory roles throughout all stages of protein synthesis. Using fluorescently-labeled components within a highly-purified in vitro translation system, we are directly characterizing structural changes of the translational machinery in real time using single-molecule Forster resonance energy transfer (smFRET) in order to elucidate the mechanisms through which these dynamics direct and regulate the individual steps of translation. Here we report new ribosome-ribosome, ribosome-tRNA, and tRNA-translation factor smFRET signals that have allowed us to fully characterize the intrinsic conformational dynamics of a ribosomal domain, the L1 stalk, as well as the coupling between L1 stalk and tRNA dynamics, throughout protein synthesis. Our data reveal that the translating ribosome can spontaneously and reversibly fluctuate between two global conformational states, and that transitions between these two states involve coupled movements of the L1 stalk and ribosome-bound tRNAs, accompanied by ratcheting of the ribosomal subunits. Furthermore, we find that elongation, release, and ribosome recycling factors uniquely recognize these global states of the ribosome and differentially affect transition rates between the two states. Thus, translation factor-mediated recognition and control over intrinsic dynamics of the ribosome plays a major mechanistic role during the elongation, termination, and recycling stages of translation. Our results support the view that specific regulation of the global state of the ribosome is a fundamental characteristic of all translation factors and a unifying theme throughout protein synthesis.