Hinge action versus grip in translocation by RNA polymerase

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 Mg2+ and also an RNA 3′-OH or a 3′-H2 affect RNAP sliding. Because RNAP grip to template suggests a sticky translocation mechanism, and because grip is enhanced by increasing K+ and Mg2+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.

An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options

“…However, the rate of transcript elongation over long DNA segments is not significantly affected by substitutions in the CRE pocket that affect pausing (54). Molecular dynamics simulations of translocation do not reveal significant occupancy of the half-translocated state (55, 56), although further application of this approach could give additional insight. Normal thermal fluctuations of the EC could allow either RNA-first or concerted RNA–DNA translocation to occur, making the half-translocated state a possible but inconsequential intermediate in the normal NAC.…”

Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

“…Pol I undergoes major conformational changes during the transcription cycle, mainly affecting the width of the DNA-binding cleft [48]. During the initiation of transcription, the cleft aperture narrows from a semi-open configuration, as seen in cryo-EM structures of the enzyme bound to Rrn3 [12,23,34], to a fully closed conformation observed in transcribing complexes [13,15] (Fig 8).…”

Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

“…Of special interest are mutants altered far from an enzyme's active site(s) that strongly affect its activity. For example, alteration of two amino acids in Thermus thermophilus RNA polymerase β' subunit located ~ 20 Ǻ from the polymerization site reduces transcription rate > 1000-fold 45 . Conversely, a single amino acid change in Saccharomyces cerevisiae RNA polymerase I subunit Rpa135, located ~ 50 Ǻ from the site of RNA synthesis, enhances transcription rate 2-to 3-fold 46 .…”

“…More extensive analyses might reveal many additional points required for long-distance communication between sites in these enzymes, as shown here for RecBCD. Co-ordination of activities of many other large enzymes, such as kinesin and ATP synthase [45][46][47] , must require communication between distant parts of the enzyme and could be similarly analyzed. RecBCD is large, has a complex reaction cycle, and is not essential for growth, making it readily amenable to mutational analysis.…”