RNA polymerase and transcription elongation factor Spt4/5 complex structure

Klein, Brianna J.; Bose, Daniel; Baker, Kevin J.; Yusoff, Zahirah M.; Zhang, Xiaodong; Murakami, Katsuhiko

doi:10.1073/pnas.1013828108

Cited by 139 publications

(168 citation statements)

References 34 publications

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“…The RNAP clamp provides binding sites for the transcription initiation factor TFE (homologous to TFIIEα) and the elongation factor Spt4/5, both of which bind competitively to the clamp coiled coil motif (15)(16)(17) of archaeal-eukaryotic RNAPs. TFE facilitates open complex formation by interacting with the RNAP clamp and stalk and the nontemplate strand (NTS) (15,18).…”

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confidence: 99%

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Schulz

Gietl

Smollett

et al. 2016

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

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Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.

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confidence: 99%

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Schulz

Gietl

Smollett

et al. 2016

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

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“…The GL was shown to interact with Escherichia coli RfaH (7), a specialized paralog of the essential transcription elongation factor NusG. Ubiquitous NusG homologs are thought to enhance elongation by bridging the βGL and the β′ clamp to stabilize the latter in a closed, pause-resistant conformation (8)(9)(10). Consistent with an important role of the GL, its sequence is relatively conserved in Bacteria (Fig.…”

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confidence: 63%

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

NandyMazumdar

Nedialkov

Svetlov

et al. 2016

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

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Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β′ clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.D uring each round of transcription, RNA polymerase (RNAP) establishes, maintains, and finally releases contacts with the DNA template and the RNA. These interactions mediate highly selective initiation, processive elongation, and precise termination and can be tuned to enable intricate regulation during the transcription cycle. RNAP resembles a crab claw in which the two pincers composed of the β′ and β subunits form an active site cleft that accommodates the nucleic acid chains (Fig. 1). The mobile β′ clamp domain that forms one of the pincers stands out as a central regulatory feature (1,2). The open clamp likely allows the loading of the promoter DNA during initiation and the release of the template and the nascent RNA during termination. The clamp is thought to close to form an active initiation complex and to remain closed during elongation but may open partially at a hairpindependent pause site (3).The clamp movements may be linked to those of the β lobe domain, which forms a part of the second pincer. The β gate loop (GL), which lies across the RNAP cleft from the tip of the clamp (Fig. 1), has been identified as an element that restricts the entry of the duplex promoter DNA into the narrow active site cleft (4), allowing only a single strand of DNA to pass through. This model posited that the DNA stands must separate outside the cleft before entry into RNAP, whereas footprinting studies demonstrated that the promoter DNA enters the active site cleft before it is opened (5). This controversy was a subject of an intense debate until a recent study revealed that the...

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“…As an extension of this point, if initiation is slow for open complexes at some promoters, retention of TFIIE in these complexes could be important to prevent inactivation. Recent work indicates that the Spt5 subunit of the DRB sensitivity-inducing factor complex should compete with TFIIE for a common binding site on the pol II clamp domain (8,15,41). Thus, loading of DRB sensitivity-inducing factor into the nascent transcription complex could also influence survival of the open complex.…”

Section: Discussionmentioning

confidence: 99%