Interrupted catalysis: the EF4 (LepA) effect on back translocation

Abstract

EF4 (LepA) promotes back translocation of tRNAs on the ribosome, and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4‐catalyzed back translocation. EF4‐dependent back translocation proceeds from post‐translocation complex (POST) to pre‐translocation complex (PRE) via a four‐step kinetic scheme, i.e., POST → I1 → I2 → I3 → PRE, that is not the simple reverse of translocation, and during which movement of the 3’‐end of peptidyl–tRNA is partially decoupled from movements of the tRNA core regions and of mRNA. EF4 may be thought of as performing an interrupted catalysis of this process, stopping at the formation of I3 rather than catalyzing the complete process of back translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I3 is likely to be important for optimizing functional protein biosynthesis. Supported by NIH grant GM071014 to B. S. C.