Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by
            <i>Escherichia coli</i>
            RNA polymerase

Davis, Caroline A.; Bingman, C.A.; Landick, Robert; Record, M. Thomas; Saecker, Ruth M.

doi:10.1073/pnas.0609888104

Cited by 87 publications

(211 citation statements)

References 39 publications

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“…In the WT λP R RPo, the NT strand T residues −10, −4, −3, and +2 were accessible to modification (Fig. 2B), consistent with previous studies (5). A different pattern was observed in the complex formed with the ΔGL RNAP: Although the −10 and +2 residues were as sensitive to modification as in the WT complex, the −4 and −3 residues were significantly protected (Fig.…”

Section: Resultssupporting

confidence: 88%

“…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 width of the RNAP cleft varies in solution and potentially is able to accommodate the duplex DNA (6).…”

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confidence: 99%

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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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Section: Resultssupporting

confidence: 88%

mentioning

confidence: 99%

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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“…The conversion of the initial closed P c complex to the initiation-ready P o complex is a multi-step process. At the λP R promoter, a competitor-resistant closed complex is a significantly accumulating kinetic intermediate (Davis et al, 2007;Record et al, 1996). Whether that is the case for basal or activated transcription at the late promoter remains to be determined, so steps 3 and 4 have been lumped together.…”

Section: Supplementary Materialsmentioning

confidence: 99%

Dissection of the Bacteriophage T4 Late Promoter Complex

Nechaev

Geiduschek

2008

Journal of Molecular Biology

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Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator gp45 is the viral replisome's sliding clamp; two sliding clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) σ subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP-sliding clamp connections differ, with only the gp33-gp45 linkage essential for activation. Formation of transcription-ready promoter complexes by the sliding clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55-gp45 but not the gp33-gp45 linkage. We interpret these findings in relation to the common model of transcriptional initiation in bacteria and highlight the roles of individual interactions of the promoter complex.

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“…1 A) (12)(13)(14). The result of these changes generates a complex in which the promoter is unwound from Ϫ11 to around ϩ3, and the protection footprint extends to around ϩ25 (9,11,(15)(16)(17)(18)(19)(20)(21). In addition, RPo is normally competitor resistant, although RPo at the very strong ribosomal promoters does not follow this rule (22,23).…”

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confidence: 99%

“…In addition, structures of , core polymerase, and holoenzyme from thermophilic bacteria (4,(24)(25)(26)(27)(28)(29) or portions of E. coli 70 (30,31) have provided 3D scaffolds on which to model these steps. Kinetic analyses using the promoter P R have revealed transcriptional intermediates in the pathway from RPc to 20, and 32 and references therein). Initially, the ds promoter DNA is thought to lie across the polymerase, making sequence-specific contacts with 70 and the ␣-CTDs.…”

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confidence: 99%

The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase

Hook-Barnard

Hinton

2009

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

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Transcription initiation is a dynamic process in which RNA polymerase (RNAP) and promoter DNA act as partners, changing in response to one another, to produce a polymerase/promoter open complex (RPo) competent for transcription. In Escherichia coli RNAP, region 1.1, the N-terminal 100 residues of 70 , is thought to occupy the channel that will hold the DNA downstream of the transcription start site; thus, region 1.1 must move from this channel as RPo is formed. Previous work has also shown that region 1.1 can modulate RPo formation depending on the promoter. For some promoters region 1.1 stimulates the formation of open complexes; at the P minor promoter, region 1.1 inhibits this formation. We demonstrate here that the AT-rich P minor spacer sequence, rather than promoter recognition elements or downstream DNA, determines the effect of region 1.1 on promoter activity. Using a P minor derivative that contains good 70 -dependent DNA elements, we find that the presence of a more GC-rich spacer or a spacer with the complement of the P minor sequence results in a promoter that is no longer inhibited by region 1.1. Furthermore, the presence of the Pminor spacer, the GC-rich spacer, or the complement spacer results in different mobilities of promoter DNA during gel electrophoresis, suggesting that the spacer regions impart differing conformations or curvatures to the DNA. We speculate that the spacer can influence the trajectory or flexibility of DNA as it enters the RNAP channel and that region 1.1 acts as a ''gatekeeper'' to monitor channel entry.

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Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Cited by 87 publications

References 39 publications

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

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

Dissection of the Bacteriophage T4 Late Promoter Complex

The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase

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Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Cited by 87 publications

References 39 publications

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

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

Dissection of the Bacteriophage T4 Late Promoter Complex

The promoter spacer influences transcription initiation via σ 70 region 1.1 of Escherichia coli RNA polymerase

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The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase