Stringent control of the RNA-dependent RNA polymerase translocation revealed by multiple intermediate structures.

Each polymerase nucleotide addition cycle is associated with two primary conformational changes of the catalytic complex: the pre-chemistry active site closure and post-chemistry translocation. While active site closure is well interpreted by numerous crystallographic snapshots, translocation intermediates are rarely captured. Here we report three types of intermediate structures in an RNA-dependent RNA polymerase (RdRP). The first two types, captured in forward and reverse translocation events, both highlight the role of RdRP-unique motif G in restricting the RNA template movement, corresponding to the rate-limiting step in translocation. By mutating two critical residues in motif G, we obtain the third type of intermediates that may mimic the transition state of this rate-limiting step, demonstrating a previously unidentified movement of the template strand. We propose that a similar strategy may be utilized by other classes of nucleic acid polymerases to ensure templating nucleotide positioning for efficient catalysis through restricting interactions with template RNA. During translocation by viral RNA-dependent RNA polymerases (RdRP), the template-product RNA duplex moves in an asymmetric manner. Here the authors describe several crystal structures of RdRP translocations intermediates that, along with enzymological data, provide a comprehensive view of RdRP translocation.