Translocation of tRNAs through the Ribosome followed by Single Molecule FRET

Abstract

Translocation of tRNAs is catalyzed by an enzyme called elongation factor G. EF-G mediated translocation is fast. For that reason it is difficult to study tRNA trajectories through the ribosome as they are affected by numerous structural rearrangements not only of the translation factor but of the whole ribosomal machinery.To monitor tRNA movement through the ribosome in real time we employ a single molecule fluorescence approach to study which pathway individual ribosomes use during translocation. We use a TIRF microscope with dual color detection to track FRET signals between ribosomes that are donor-labeled on protein L11 and carry acceptor-labeled tRNA molecules. To slow down translocation to a rate amenable by our TIRF microscope we have used a mutant of EF-G.Our experiments reveal an intermediate state of translocation that has not been observed before. This intermediate is in equilibrium with pre and post states and is consumed in the course of translocation to form the post translocation state. The transition of the intermediate to the post state indicates that translocation is driven to completion by an irreversible step taking place after the tRNAs have arrived in their post translocation positions. We suggest that the irreversible step is the dissociation of either EF-G or the E site tRNA from the ribosome.