Argonaute (Ago) proteins are the key component of the RNA-induced silencing complex and mediate RNA interference (RNAi) in association with small RNAs. Although overall the mechanism of RNAi is well understood, many molecular details of this complex process are not. Here we report about in-depth steady-state and, in particular, pre-steady-state characterization of siRNA binding, target RNA recognition, sequence-specific cleavage and product release by recombinant human Ago 2 (hAgo2). In combining our biochemical studies with crystal structures of bacterial Ago proteins and of recently released hAgo2, we relate kinetic data to conformational changes along the pathway and propose a comprehensive minimal mechanistic model describing fundamental steps during RNAi. Furthermore, in contrast to the current conception, our hAgo2 preparations are programmable with double-stranded siRNA. Accordingly, the system investigated represents a functional minimal RNA-induced silencing complex.
Keywords: enzyme kinetics; fluorescence spectroscopy; gene silencing; pre-steady-state kinetics.