Unveiling translocation intermediates of RNA polymerase.

Translocation of RNA polymerase (RNAP) is a robust target for regulation of gene expression in prokaryotes and eukaryotes (1–3). During elongation, RNAP frequently encounters a broad range of DNA/RNA conformations (RNA hairpins, curved and cruciform DNA), DNA-binding proteins, DNA lesions, and misincorporation events at the 3′ ends of the RNA. These encounters impede forward translocation, leading to RNAP pausing (3). There are many protein factors that strengthen or weaken pausing by targeting translocation, such as archaeal/eukaryotic Spt5 and bacterial NusG/RfaH (4) or N/Nun proteins of lambdoid phages (3). In metazoa, RNAP II pausing in promoter-proximal regions plays a role in having polymerases in place for a rapid transcription response to environmental stimuli, such as heat-shock or cell differentiation, and maintaining a basal level of gene expression (5, 6). Translocation blocks also initiate RNAP backtracking (7). Backtracking events disengage the 3′ RNA end from the RNAP catalytic site, which stabilizes pausing; this type of event broadly controls gene transcription in eukaryotes (8). Forward translocation of RNAP along DNA has long been regarded as the movement of the RNA–DNA hybrid through the catalytic cleft, which vacates the active center, termed i+1 site, for an NTP binding. In this sense, the mechanism of translocation has been primarily focused on the hybrid movement, with only limited emphasis on the DNA sequences surrounding the hybrid (1, 2, 9). However, several reports indicate that the DNA sequences immediately upstream and downstream from the hybrid regulate translocation (7, 10–12). In PNAS, Silva et al. (13) identify an important component of the translocation mechanism using millisecond molecular dynamics (MD) simulation of translocation of yeast RNAP II: Translocation of the hybrid occurs before entry of the template DNA base to the i+1 site, where it can pair with an NTP. A similar scenario had been suggested based on the X-ray structure of RNAP II with the transcription inhibitor α-amanitin (9). The MD simulation posits that the entering of the template DNA base requires its stacking interaction with Tyr836 in the middle of the bridge helix (BH), a long α-helix separating the i+1 site from the downstream DNA (Fig. 1A). The Tyr residue in the BH is highly conserved from Escherichia coli to human. The bent and straight forms of the BH have been previously observed in the crystal structure of bacterial RNAP and eukaryotic RNAP II (14, 15), leading to one hypothesis that the bent-stretch transition or oscillation of the BH coupled with the movement of the trigger loop, another flexible part of the i+1 site involved in catalysis and substrate binding (16), is a driving force for forward translocation by forming a “pawl” (17). By revealing long-time translocation dynamics with atomic resolution, Silva et al. (13) show that the stacking interaction of the i+1 DNA base with the tyrosine of the BHmay form an additional pawl. Based on kinetic modeling, Silva et al. argue that the template DNA base–Tyr836 stacking generates a metastable intermediate, which decreases the high activation energy of the transition state for forward translocation (13). The MD simulation using in silico Y836V mutant lacking the aromatic side chain of tyrosine showed interrupted translocation, supporting this hypothesis. Although this argument is conceivable, it remains uncertain whether each translocation intermediate in all sequence contexts has a homogeneous state that is separated by the activation energy or the energy barrier >> 0.5 kBT, the averaged value of thermal energy per degree of freedom, where kB is the Boltzmann constant and T is the absolute temperature. This assumption is required A