howing the in and out conformations of the residue 288–292 loop. The side view shows how the ribose of the −1 position
template strand nucleotide (magenta) makes direct contact with the loop in the in conformation but has a clear steric clash
when the loop is in the out conformation. The top view shows the direction and magnitude of the loop movement relative to
the active site, nascent base pair, and ddCTP base paired to the templating +1 site nucleotide. (B) Kinetic scheme of the
polymerase elongation cycle illustrating the model for the different steps involving conformational changes in the loop. The
EC states reflect the six-state cycle based on the picornaviral polymerase EC structures [8,21]. The flip of Ser288 from up
to down is a pre-catalytic transition involved in NTP repositioning while the movement of the loop from in to out is
associated with the post-catalysis translocation step. Our finding that hydrophobic residues at position 290 increase
polymerase activity while polar residues reduce activity further suggests that the viral RdRPs may have a distinct sixth step
following pyrophosphate release whereby residue 290 is reinserted into its pocket, returning the loop to the in/up
conformation and resetting the active site for the next round of catalysis.