Bridge helix and trigger loop perturbations generate superactive RNA polymerases

Tan, Lin; Wiesler, Simone C.; Trzaska, Dominika; Carney, Hannah C.; Weinzierl, Robert O. J.

doi:10.1186/jbiol98

Cited by 85 publications

(163 citation statements)

References 49 publications

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“…4C). These results are consistent with mutagenesis experiments on a related multisubunit RNAP (40), where valine mutants at this position have lower transcription activity compared with phenylalanine and WT (with tyrosine).…”

Section: The Free-energy Landscape Suggests a Single Rate Limiting Stsupporting

confidence: 89%

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: The Free-energy Landscape Suggests a Single Rate Limiting Stsupporting

confidence: 89%

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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“…Substitution of this region in Taq RNAP with the corresponding region from Dra RNAP resulted in a significant increase in the rate of nucleotide addition, especially at low and moderate temperatures. Remarkably, the observed increase was much more dramatic than reported previously for substitutions in TL and BH that stimulated the rate of elongation 2-to 3-fold (10,14). We also showed that substitutions of two individual residues from this region in Taq RNAP, Q1046A and S1074A, increased the rate of catalysis about 3-fold.…”

Section: Discussionsupporting

confidence: 57%

“…Even single amino acid substitutions in the structural elements involved in catalysis dramatically alter the catalytic properties of RNAP (3,9,12,14,17,18). We thus wondered whether the differences in the cold sensitivity of the Taq and Dra enzymes could be conferred by changes of just a few residues.…”

Section: Resultsmentioning

confidence: 99%

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Allosteric control of catalysis by the F loop of RNA polymerase

Miropolskaya

Artsimovitch

Klimašauskas³

et al. 2009

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

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Bacterial RNA polymerases (RNAPs) undergo coordinated conformational changes during catalysis. In particular, concerted folding of the trigger loop and rearrangements of the bridge helix at the RNAP active center have been implicated in nucleotide addition and RNAP translocation. At moderate temperatures, the rate of catalysis by RNAP from thermophilic Thermus aquaticus is dramatically reduced compared with its closest mesophilic relative, Deinococcus radiodurans. Here, we show that a part of this difference is conferred by a third element, the F loop, which is adjacent to the N terminus of the bridge helix and directly contacts the folded trigger loop. Substitutions of amino acid residues in the F loop and in an adjacent segment of the bridge helix in T. aquaticus RNAP for their D. radiodurans counterparts significantly increased the rate of catalysis (up to 40-fold at 20°C). A deletion in the F loop dramatically impaired the rate of nucleotide addition and pyrophosphorolysis, but it had only a moderate effect on intrinsic RNA cleavage. Streptolydigin, an antibiotic that blocks folding of the trigger loop, did not inhibit nucleotide addition by the mutant enzyme. The resistance to streptolydigin likely results from the loss of its functional target, the folding of the trigger loop, which is already impaired by the F-loop deletion. Our results demonstrate that the F loop is essential for proper folding of the trigger loop during nucleotide addition and governs the temperature adaptivity of RNAPs in different bacteria.nucleotide addition ͉ RNA cleavage ͉ streptolydigin ͉ temperature adaptation ͉ transcription C ellular multisubunit RNA polymerases (RNAPs) transcribe genes of up to tens of thousands nucleotides long with high precision and processivity. The catalytic cycle of RNAP is driven by complex conformational changes that accompany NTP binding, catalysis, and RNAP translocation. Recent studies identified two elements in the largest RNAP subunit (␤Ј in bacteria, Rpb1 in eukaryotic RNAP), the trigger loop (TL, or G loop), and the bridge helix (BH, or F-bridge helix), which appear to play the key role during the nucleotide addition cycle (Fig. 1). In the absence of a nucleotide substrate, the TL [amino acids 1234-1254 in Thermus aquaticus (Taq) and Thermus thermophilus RNAPs; amino acids 1077-1097 in Saccharomyces cerevisiae (Sce) RNAPII] adopts an open conformation in which its central part is unstructured (1, 2). Binding of an NTP substrate induces folding of the TL, resulting in extension of the two ␣-helixes at the base of the TL and creating a closed, catalytically competent conformation of the active center in which the NTP is properly aligned with the 3Ј-OH of the nascent RNA for facile catalysis (Fig. 1 A-C) (3, 4). The folded TL and the BH form a three-helix bundle that interacts with the substrate NTP and the template DNA base. In particular, the TL residues M1238 and H1242 (Taq numbering is used throughout the manuscript unless otherwise indicated) contact the base and the phosphate moieties of the ...

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“…This provided us with a powerful tool that is readily accessible to saturation mutagenesis approaches during which we can study protein-protein interactions in an unbiased manner and on a single-residue level. Saturation mutagenesis has given us extensive insight into the protein dynamics accompanying the nucleotide addition cycle catalysed by mjRNAP that structural data have failed to uncover (29,30). Given the success in this system, we recently included the interface of the mjTFIIB linker and mjRNAP into our studies to further investigate the stimulation effect of the linker on mjRNAP activity (26).…”

Section: The Tfiib/rnap Interface: Advantages Of Biochemical Analysismentioning

confidence: 99%

Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase

Wiesler

Werner

Weinzierl

2013

Methods in Molecular Biology

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Several co-crystals of the TFIIB-RNAP II interface exist suggesting the TFIIB linker region to penetrate the active centre, yet the understanding of the resulting protein-protein interactions and their impact on the transcription cycle has remained elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates and measuring the phosphodiester bond formation they catalyse. These assays can be used to measure the effect of transcription factors and mutations on the rate of the catalytic reaction. Notably, they have served to reveal and characterise a significant stimulation activity of TFIIB on RNAP that was previously unknown.

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Bridge helix and trigger loop perturbations generate superactive RNA polymerases

Cited by 85 publications

References 49 publications

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Millisecond dynamics of RNA polymerase II translocation at atomic resolution

Allosteric control of catalysis by the F loop of RNA polymerase

Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase

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