Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Abstract

Transcription is a central step in gene expression, in which the DNA template is processively read by RNA polymerase II (Pol II), synthesizing a complementary messenger RNA transcript. At each cycle, Pol II moves exactly one register along the DNA, a process known as translocation. Although X-ray crystal structures have greatly enhanced our understanding of the transcription process, the underlying molecular mechanisms of translocation remain unclear. Here we use sophisticated simulation techniques to observe Pol II translocation on a millisecond timescale and at atomistic resolution. We observe multiple cycles of forward and backward translocation and identify two previously unidentified intermediate states. We show that the bridge helix (BH) plays a key role accelerating the translocation of both the RNA:DNA hybrid and transition nucleotide by directly interacting with them. The conserved BH residues, Thr831 and Tyr836, mediate these interactions. To date, this study delivers the most detailed picture of the mechanism of Pol II translocation at atomic level.Markov state model | molecular dynamics | trigger loop T he RNA polymerase is the central component of gene expression in all living organisms, transferring genetic information from DNA to RNA. In eukaryotes, the RNA polymerase II (Pol II) enzyme is responsible for transcribing DNA into messenger RNA. In the past decade, a number of X-ray crystallographic structures of Pol II have been obtained at different stages of the transcription process, providing a static picture of how this complex machine performs its function (1, 2). Transcription is a multistep process consisting of initiation, elongation, and termination, where elongation is composed of consecutive nucleotide addition cycles (NACs). In each NAC, the NTP substrate first diffuses into Pol II active site through the secondary channel (3-5) or alternatively the main channel (6). Upon correct NTP binding to the Pol II active site, the trigger loop (TL) conformation switches from an inactive open state to an active closed state (7). The closure of the active site subsequently facilitates the catalysis of the nucleotide addition reaction (7), followed by release of the pyrophosphate ion (PPi). To proceed to the next NAC, Pol II must translocate from a pretranslocation state, in which the active site is still occupied by the newly added nucleotide at 3′-RNA, to a posttranslocation state. During translocation, the template DNA and RNA must move by exactly one register, once again creating a free insertion site (i site) (1,2,5,(8)(9)(10)(11)(12)(13).Although static snapshots of X-ray structures of Pol II pretranslocation and posttranslocation states are valuable, the dynamics underlying the fundamental RNA polymerase translocation mechanism remain poorly understood (14). Two models of translocation have been proposed based on structural, biochemical, and genetic approaches. On one hand, for single subunit T7 RNAP, PPi release is suggested to be mechanically coupled to the opening motion of the O-helix (co...

Unveiling translocation intermediates of RNA polymerase

“…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.…”

“…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 BH may form an additional pawl.…”

“…However, several reports indicate that the DNA sequences immediately upstream and downstream from the hybrid regulate translocation (7,(10)(11)(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).…”

“…This assumption is required before one can use rate equations for modeling RNAP translocation (3,18). For the same reason, applying the Markov process to construct the millisecond-long MD simulation of translocation needs additional validation (13). In the Markov process, probability distribution of the position of the reactant molecule at the time does not depend on the previously visited states.…”

“…First, the former indicated that translocation and pyrophosphate release occur at ∼10 μs and ∼1 μs, respectively (13,23). However, a biochemical study of E. coli RNAP revealed that both processes occur around 10 ms (24).…”