Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

Kireeva, Maria L.; Opron, Kristopher; Seibold, Steve A.; Domecq, Céline; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Burton, Zachary F.

doi:10.1186/2046-1682-5-11

Cited by 37 publications

(86 citation statements)

References 54 publications

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“…This Gly-Gly pair is referred to as a "hinge," and we found that it can transiently lose its α-helix secondary structure (Fig. S5C), consistent with previous MD simulation studies on a short timescale (21). We also examined if the BH bending is coupled with the clamp motion by computing its cross-correlation (Fig.…”

Section: Resultsmentioning

confidence: 63%

“…Our simulations of translocation events, together with previous simulations (9,(20)(21)(22), provide important mechanistic and kinetic insights of several key steps in Pol II transcription. Our previous simulation study has suggested that the PPi release is rapid [at ∼1 μs (9)].…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, previous all-atom MD simulation studies have provided valuable insights into the dynamics of Pol II (9,(20)(21)(22). 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.…”

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confidence: 99%

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Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Silva

Weiss

Avila

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

144

199

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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...

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Section: Resultsmentioning

confidence: 63%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Silva

Weiss

Avila

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

144

199

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“…In these conformations, during translocation, it is the BH that is seen to play a more prominent role [as it was originally proposed by Bar-Nahum et al (4)] and the TL, although unfolded in both cases, is thought to assist the BH in translocation. As a whole, structural studies indicate that transcription elongation most likely involves a concerted motion of the "BH/TL unit" (9,14,16,(19)(20)(21)(22).In addition, several bulk studies revealed that most TL mutations that alter RNAP's elongation rate affect its pausing behavior too (4,8,10), suggesting that the TL is also involved in polymerase pause regulation. Furthermore, Toulokhonov et al (8) have shown that in paused complexes the TL adopts a conformation that inhibits nucleotide addition, but which also blocks binding of the transcription inhibitor streptolydigin (known to bind the unfolded TL).…”

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confidence: 99%

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confidence: 99%

Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Mejia

Nudler

Bustamante

2014

Proc. Natl. Acad. Sci. U.S.A.

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Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP's pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity. RNA polymerase (RNAP) has been the subject of study for almost five decades by means of a large array of techniques. In the last decade, crystallographic structures of the bacterial and eukaryotic polymerase have allowed researchers to obtain snapshots of the conformational changes that occur deep inside the enzyme, near its catalytic center. Based on these structures, an element, the F-bridge or bridge helix (BH), was first hypothesized to be the essential component in the translocation mechanism of RNAP (1-4). Later, crystal structures of the full elongation complex [RNA, DNA, and nucleoside triphosphate (NTP) bound] identified another structure in close proximity to the BH, termed the trigger loop (TL) (5-7), as another important element in the translocation mechanism. This structure was seen to adopt distinct conformations during the catalytic process, suggesting its role in the kinetic cycle of RNAP. In particular, the TL was seen to contain a dynamic domain that undergoes an unfolding transition during transcription (here termed the TLtip) and another which remains helical throughout the cycle known as the TL base helices. These observations prompted further biochemical characterization of these two structures in their WT form and in variety of point mutants of the BH and TL elements (4,(8)(9)(10)(11)(12).The high-resolution structure of an elongation complex of the Thermus thermophilus polymerase (13) shows the TL in a fully folded helix-turn-helix structure and in close contact with the BH (Fig. S1 A-C). This conformation, in which the TL blocks the secondary channel and correctly positions the incoming NTP for incorporation to occur, has been termed "closed" (10, 14). Based on these structures and other biochemical studies (4,7,8,(15)(16)(17)(18) it has been proposed that NTP binding and the resulting folding of the TL (with its corresponding interactions with the BH) serves as the pawl that rectifies the polymerase's Brownian oscillations on the template by not allowi...

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Dynamics of bridge helix bending in RNA polymerase II

Wang

et al. 2017

Proteins

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One of critical issues for RNA polymerase is how the enzyme translocates along the DNA substrate during transcription elongation cycle. Comparisons of the structure of RNA polymerase II (Pol II) with that of bacterial enzyme have suggested that the transition of the bridge helix (BH) from straight to flipped-out conformations facilitates the translocation of upstream DNA-RNA hybrid. However, the flipped-out conformation of BH in Pol II has not been observed up to now and the detailed mechanism of how the BH facilitating upstream hybrid translocation still remains obscure. Here we use all-atom molecular dynamics simulations to study the transition dynamics of BH in Pol II. Two different flipped-out conformations (termed as F1 and F2) are derived from our simulation trajectories, both of which could contribute to upstream hybrid translocation. In particular, the structure of BH in F2 conformation shows nearly identical to that observed in free bacterial enzyme, showing the existence of the flipped-out conformation in Pol II. Analysis of hydrogen bonds and salt bridge formed intra BH in different conformations indicates that the flipped-out conformations are more unstable than the straight conformation. Moreover, a detailed understanding of how the transition of BH conformations facilitating upstream hybrid translocation is given. Proteins 2017; 85:614-629. © 2016 Wiley Periodicals, Inc.

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Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

Cited by 37 publications

References 54 publications

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

Dynamics of bridge helix bending in RNA polymerase II

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