Interrupted Catalysis: The EF4 (LepA) Effect on Back-Translocation

Abstract

EF4, although structurally similar to the translocase EF-G, 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 and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST→I1→I2→I3→PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3′-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, 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.