Active site opening and closure control translocation of multisubunit 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 BH may form an additional pawl.Based on kinetic modeling, Silva et al. argue that the template ...

supporting

confidence: 66%

Unveiling translocation intermediates of RNA polymerase

Imashimizu

Kashlev

2014

“…The extra translocation step is responsible for the slow catalytic rate of the minor fraction of TEC A 11 . It has been shown recently that forward RNAP translocation is a rate-limiting step at some DNA sequences (7,17,39). Nun converted the double-exponential curve to a singleexponential curve, presumably by inactivating the minor pretranslocated fraction (compare the black and red curves in Fig.…”

Section: Effect Of Nun-resistant Rnap Mutants and A Nonfunctional Nunmentioning

confidence: 92%

mentioning

confidence: 97%

“…Restriction of this transition in hybrid length may hinder forward translocation and induce subsequent pausing and/or arrest. Although a conventional view suggests that translocation is rapid and reversible (15), and occurs in both directions with a rate substantially faster than catalysis, recent data argue that, at least at some sequences, RNAP dwells primarily in the posttranslocated state with a slow return to the pretranslocated state (16,17).NusG is the only known elongation factor that promotes forward translocation; factors that directly restrict translocation in both directions have yet to be reported (6). It has been suggested that Nun may inhibit transcription elongation by blocking RNAP translocation (8).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.T ranscription elongation is highly processive, yet the rate of nucleotide addition varies significantly for different ternary elongation complexes (TECs). In the normal elongation pathway, each nucleotide addition is followed by translocation of RNA polymerase (RNAP) along DNA in single-nucleotide increments. This process transfers the RNA 3′ end from the i + 1 site to the i site of the active center, thus allowing binding of the next NTP and subsequent phosphodiester bond formation. Translocation is thought to be a stochastic, rapid, and fully reversible process and not rate-limiting for elongation (1, 2). Instead, NTP sequestration, phosphodiester bond formation, or pyrophosphate release has been suggested to be rate-limiting for transcription elongation (3, 4). However, several reports strongly suggest that translocation may be at least partially rate-limiting for elongation (5-9).Immediately following bond formation, a catalytically inactive and highly pyrophosphorolytic (pretranslocated) state is formed. In this state, the 3′ RNA end remains in the i + 1 site of the active center. The 3′ RNA thus prevents NTP binding and generates a substrate for pyrophosphorolysis. To bind the next NTP, RNAP must translocate 1-bp forward along the DNA register to form a catalytically active and pyrophosphate-resistant (posttranslocated) state. Stationary RNAP has been suggested to "ratchet" between the two states via Brownian motion (5). NTP bound to the transient posttranslocated state acts as a pawl that interferes with backward translocation, thereby favoring the formation of a phosphodiester bond. Retention of this NTP in the posttranslocated TEC is facilitated by isomerization of the active site that blocks substrate exit, and aligns it for catalysis (10, 11)...

“…However, it is important to point out that previous MD simulations are limited to a few hundred nanoseconds, at which proteins may only undergo local conformational changes such as side-chain rotations and loop motions. These MD simulations fall far short of biologically relevant timescales of Pol II translocation (tens of microseconds to millisecond or even longer) (23,24). Directly simulating the millisecond timescale of a huge system like Pol II in explicit solvent (nearly half a million atoms)…”

mentioning

confidence: 99%

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Silva

Weiss

Avila

et al. 2014

144

199

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...