Abstract
Based on molecular dynamics simulations and functional studies, a conformational mechanism is posited for forward translocation by RNA polymerase (RNAP). In a simulation of a ternary elongation complex, the clamp and downstream cleft were observed to close. Hinges within the bridge helix and trigger loop supported generation of translocation force against the RNA-DNA hybrid resulting in opening of the furthest upstream i-8 RNA-DNA bp, establishing conditions for RNAP sliding. The β flap tip helix and the most N-terminal β' Zn finger engage the RNA, indicating a path of RNA threading out of the exit channel. Because the β flap tip connects to the RNAP active site through the β subunit double-Ψ-β-barrel and the associated sandwich barrel hybrid motif (also called the flap domain), the RNAP active site is coupled to the RNA exit channel and to the translocation of RNA-DNA. Using an exonuclease III assay to monitor translocation of RNAP elongation complexes, we show that K(+) and Mg(2+) and also an RNA 3'-OH or a 3'-H(2) affect RNAP sliding. Because RNAP grip to template suggests a sticky translocation mechanism, and because grip is enhanced by increasing K(+) and Mg(2+)concentration, biochemical assays are consistent with a conformational change that drives forward translocation as observed in simulations. Mutational analysis of the bridge helix indicates that 778-GARKGL-783 (Escherichia coli numbering) is a homeostatic hinge that undergoes multiple bends to compensate for complex conformational dynamics during phosphodiester bond formation and translocation.