Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Vvedenskaya, Irina O.; Vahedian-Movahed, Hanif; Zhang, Yuanchao; Taylor, Deanne; Ebright, Richard H.; Nickels, Bryce E.

doi:10.1073/pnas.1603271113

Cited by 40 publications

(35 citation statements)

References 35 publications

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“…The discriminator region between the −10 hexamer and the transcription start site (TSS; +1) has recently emerged as an important modulator of the open complex properties (11)(12)(13)(14)(15)(16)(17)(18). In a stable open complex formed by Thermus thermophilus RNAP, the −6 and −5 bases of the discriminator NT strand (NT DISC ) make crucial contacts with σ1.2, and βArg371 (E. coli numbering) interacts with the adjacent −4 and −3 bases (14).…”

Section: Resultsmentioning

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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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: Resultsmentioning

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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“…Promoter recognition is thought to be mediated by sigma factor-DNA interaction and core promoter elements comprising Ϫ35, Ϫ10, and extended Ϫ10 regions (12,23); however, a high-resolution study of an open promoter complex with Thermus thermophilus holoenzyme recently revealed the presence of a new determinant called the core recognition element (CRE) that is thought to provide sequence-specific promoter recognition of the Ϫ4 to ϩ2 region of the nontemplate DNA strand (89). Additional studies in E. coli have extended these findings and shown that the CRE can modulate transcription start site selection, elongation, and pausing and that mutation of the CRE impairs transcription (88,97).…”

Section: Discussionmentioning

confidence: 99%

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

et al. 2016

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Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported ⌬spx strain harbored suppressor mutations that allowed it to grow without spx. One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [rpoB-P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx. The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the ⌬spx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB-P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx. Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus, including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism. IMPORTANCEThe survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus, including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target. Staphylococcus aureus is a major human pathogen that causes a diversity of disease ranging from relatively minor to invasive and systemic disease with significant morbidity and mortality (1-7). S. aureus is common both in the community and in hospital environments and has the ability to transiently colonize the skin and mucous membranes of most humans,...

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“…MASTER has been used to define, comprehensively, critical sequence determinants for transcription start site selection, reiterative transcription initiation, and transcript yields for E. coli RNAP (Winkelman et al, 2016; Vvedenskaya et al, 2016; Vvedenskaya et al, 2015; Hochschild, 2015). …”

Section: Discussionmentioning

confidence: 99%

“…In addition, for a given promoter sequence variant, the sequence of RNA 5' ends generated from the sequence variant defines the positions where transcription start site selection occurs for this promoter sequence [see (Vvedenskaya et al, 2018; Vvedenskaya et al, 2016; Vvedenskaya et al, 2015; Winkelman et al, 2016)] and can be used to define the extent of reiterative transcription initiation (“slippage synthesis”) for this promoter sequence (Vvedenskaya et al, 2015)…”

Section: High-throughput Sequencing Of Rna 5' Ends (5' Rna-seq)mentioning

confidence: 99%

Analysis of Bacterial Transcription by “Massively Systematic Transcript End Readout,” MASTER

Vvedenskaya

Goldman

Nickels

2018

Methods in Enzymology

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A systems-level view of cellular gene expression requires understanding the mechanistic principles governing each step of transcription. In this chapter, we describe a massively-multiplexed method for the analysis of the relationship between nucleic-acid sequence and transcription termed “MASTER,” for massively systematic transcript end readout. MASTER enables parallel measurements of transcription output from at least 410 (~1,000,000) individual template sequences in vitro and in vivo. MASTER involves constructing a DNA template library of barcoded sequences, generating RNA transcripts from the library during transcription in vitro or in vivo, and analyzing the relative abundance and 5'-end sequences of the RNA transcripts by high-throughput sequencing. MASTER provides a powerful, rapid, and versatile method to identify sequence determinants of each step of transcription and to define the mechanistic basis by which these sequence determinants dictate transcription output.

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Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Cited by 40 publications

References 35 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

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

Analysis of Bacterial Transcription by “Massively Systematic Transcript End Readout,” MASTER

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