Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues

Xu, Liang; Butler, Kyle V.; Chong, Jenny; Wengel, Jesper; Kool, Eric T.; Wang, Dong

doi:10.1093/nar/gku238

Cited by 18 publications

(23 citation statements)

References 49 publications

(64 reference statements)

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“…For the 3′-5′ wild-type system, we expected the TL to be closed during nucleotide incorporation and therefore the catalytic rate would sharply decrease with α-amanitin treatment. Indeed, the observed rate constant drops to 9.3 ± 0.4 min −1 , with an ∼80-fold decrease, which is consistent with the reported results (29,33). Strikingly, the 2′-5′ linkage alteration in the primer does not affect the TL conformation because there is an over 90-fold decrease in the rate constant, from 12 ± 1 min −1 to 0.13 ± 0.01 min −1 , with α-amanitin treatment (Fig.…”

Section: Resultssupporting

confidence: 92%

“…(29,31,32). Recently, we have used α-amanitin and synthetic nucleotide analogs to profile the TL conformations (33). As aforementioned, the 2′-5′ linkage alteration at either primer or template strand greatly affects the capacity of pol II for nucleotide selection and incorporation.…”

Section: Resultsmentioning

confidence: 99%

“…In these assays, we used high concentration of α-amanitin (500 mg/L) to make sure that the trigger loop of pol II was fully inhibited (29,33). Briefly, the transcriptional scaffolds were preincubated with a fourfold amount of pol II at room temperature for 5 min before addition of α-amanitin.…”

Section: Methodsmentioning

confidence: 99%

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Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Zhang

Chong

et al. 2014

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

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Nonenzymatic RNA polymerization in early life is likely to introduce backbone heterogeneity with a mixture of 2′-5′ and 3′-5′ linkages. On the other hand, modern nucleic acids are dominantly composed of 3′-5′ linkages. RNA polymerase II (pol II) is a key modern enzyme responsible for synthesizing 3′-5′-linked RNA with high fidelity. It is not clear how modern enzymes, such as pol II, selectively recognize 3′-5′ linkages over 2′-5′ linkages of nucleic acids. In this work, we systematically investigated how phosphodiester linkages of nucleic acids govern pol II transcriptional efficiency and fidelity. Through dissecting the impacts of 2′-5′ linkage mutants in the pol II catalytic site, we revealed that the presence of 2′-5′ linkage in RNA primer only modestly reduces pol II transcriptional efficiency without affecting pol II transcriptional fidelity. In sharp contrast, the presence of 2′-5′ linkage in DNA template leads to dramatic decreases in both transcriptional efficiency and fidelity. These distinct effects reveal that pol II has an asymmetric (strand-specific) recognition of phosphodiester linkage. Our results provided important insights into pol II transcriptional fidelity, suggesting essential contributions of phosphodiester linkage to pol II transcription. Finally, our results also provided important understanding on the molecular basis of nucleic acid recognition and genetic information transfer during molecular evolution. We suggest that the asymmetric recognition of phosphodiester linkage by modern nucleic acid enzymes likely stems from the distinct evolutionary pressures of template and primer strand in genetic information transfer during molecular evolution.2′-5′ phosphodiester linkage | trigger loop | synthetic nucleic acid analogues T he remarkable capacity of RNA as the functional catalyst as well as the genetic information carrier provides a strong support for the RNA world hypothesis, in which RNA is proposed to play a key role in the early evolution of primitive life before DNA and protein (enzyme) evolved (1, 2). One critical issue for RNA replication in early life is backbone heterogeneity because RNA contains both 2′-OH and 3′-OH, in which both can form phosphodiester bond during RNA polymerization. Indeed, previous studies revealed that nonenzymatic RNA replication would lead to a mixture of 2′-5′ and 3′-5′ linkages (Fig. 1A) (3-9). The 2′-5′-linked nucleic acids can form duplex structures by themselves or by pairing with natural nucleic acids (10-15). The 2′-5′ linkage substitutions in some functional RNAs retain molecular recognition and catalytic properties (16)(17)(18)(19). These discoveries suggested that the 2′-5′ linkage in nucleic acids could be an important alternative linkage during early evolution.This backbone heterogeneity problem is largely eliminated during evolution. Modern nucleic acids are dominated by 3′-5′ linkages. The 2′-5′ RNA linkages are only present in a few specific processes. One example is the formation of lariat RNA intron during splicing, where the 2′-OH of an...

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Zhang

Chong

et al. 2014

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

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“…However, in view of our previous results, these observations can be rationalized if the analogs modulate the rate of elongation in a manner directly proportional to the stability conferred by these nucleotides to the hybrid through contacts that favor the folding of the TL (39). Indeed, in support of this interpretation, all values of velocity and pause frequency, in the presence and absence of analogs and for the mutant and WT enzymes, form a well-defined trend, suggesting that both conditions affect the same kinetic rate ( Fig.…”

Section: Tl Foldingmentioning

confidence: 78%

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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“…During transcription, Pol II moves along and recognizes the DNA template strand, and selects the matched nucleotide triphosphate (NTP) for synthesizing complementary RNA transcripts with a very low error rate (less than 0.001%) (Imashimizu et al, 2014; Kaplan, 2013; Liu, Bushnell, and Kornberg, 2013; Martinez-Rucobo and Cramer, 2013; Svejstrup, 2013; Svetlov and Nudler, 2013; Xu et al, 2014; Zhang and Wang, 2013). Pol II active site is highly evolved to select for the cognate substrate that matches the DNA template through a major conformational change of trigger loop to seal the active site and form an extensive interaction network with cognate substrate poised for nucleotide addition and to exclude non-cognate substrates (Kaplan, Larsson, and Kornberg, 2008; Kellinger et al, 2012; Kireeva et al, 2008; Wang et al, 2006; Yuzenkova et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

Wang

Chong

et al. 2015

Critical Reviews in Biochemistry and Molecular Biology

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Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation.

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Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues

Cited by 18 publications

References 49 publications

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

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

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

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