Mapping σ54-RNA Polymerase Interactions at the –24 Consensus Promoter Element

Burrows, Patricia C.; Severinov, Konstantin; Ishihama, Akira; Buck, Martin; Wigneshweraraj, Sivaramesh

doi:10.1074/jbc.m303596200

Cited by 30 publications

(58 citation statements)

References 56 publications

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“…This is because nucleotides Ϫ78 to Ϫ63 upstream of the rpoS ORF comprise a theoretical, RpoN-dependent consensus Ϫ24/Ϫ12 promoter (33,34). Many studies with various bacteria have indicated close contact of RpoN with such a Ϫ24/Ϫ12 promoter region (5,6,21,33). However, as yet, there have been no experimental data to substantiate this prediction for B. burgdorferi.…”

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confidence: 77%

Evidence that RpoS (σ ^S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ ⁵⁴ /σ ^N )

et al. 2007

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The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.Lyme disease, caused by Borrelia burgdorferi, is the most commonly reported arthropod-borne disease in both the United States and Europe (32). At the molecular level, certain membrane lipoproteins of B. burgdorferi are vital for maintaining the spirochete in its zoonotic transmission cycle between ticks and mammals. For example, the reciprocal regulation of outer surface (lipo)proteins A (OspA) and C (OspC) is the best-studied paradigm of the dramatic alterations in protein expression patterns that ensue as the spirochete transitions from the arthropod vector into mammalian tissues (8,22,30,31). Our lab determined previously that expression of OspC is regulated by the alternative sigma factor RpoS ( S ) (15, 40). RpoS, though, must first be activated by another alternative transcription factor, RpoN ( 54 / N ), resulting in an RpoNRpoS regulatory network (10, 15). However, the precise mechanism(s) by which RpoN activates rpoS has not been determined. RpoN could control rpoS expression directly by binding to a region near the rpoS open reading frame (ORF). Alternatively, another transactivator induced by RpoN might activate rpoS expression.We have been most attracted to the notion that RpoN binds directly to a region upstream of the rpoS ORF to facilitate RpoS expression in B. burgdorferi. This is because nucleotides Ϫ78 to Ϫ63 upstream of the rpoS ORF comprise a theoretical, RpoN-dependent consensus Ϫ24/Ϫ12 promoter (33, 34). Many studies with various bacteria have indicated close contact of RpoN with such a Ϫ24/Ϫ12 promoter region (5, 6, 21, 33). However, as yet, there have been no experimental data to substantiate this prediction for B. burgdorferi. The purpose of this study was to garner additional evidence that would either substantiate or refute the hypothesis that rpoS is regulated directly by RpoN.Identification of rpoS initiation of transcription. Determination of the initiation of transcription for a given gene can provide strategic information for identifying a gene's nearby promoter. rpoS transcripts of B. burgdorferi BbAH130 (41) were reverse transcribed in BD SMART-RACE (switching mechanism at 5Ј end of RNA transcript-rapid amplification of cDNA ends) reactions (BD Biosciences, San Jose, CA) using two rpoS-specific primers (rpoSR422 and rpoSR232) (Table 1), which are 422 and 232 bases, respectively, downstream of the rpoS translation start site. The BD SMART IIA primer, which anneals to the BD SMART IIA oligonucleotide linked to the 3Ј end of the first-strand cDNA, and an rpoS-specific primer upstream of the gene-specific primers used in first-strand cDNA synthesis (rpoSR125) ( Table 1) were used to amplify the resulting cDNAs. PCR-amplified inserts clo...

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confidence: 77%

Evidence that RpoS (σ ^S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ ⁵⁴ /σ ^N )

et al. 2007

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“…This can explain why ⌬1149 -1190 E 54 has reduced activity for closed complex formation. Because DNA contacts within the closed complex are exclusively made by 54 (17,19), 4 we propose that deletion of ␤Ј residues 1149 -1190 leads to repositioning of 54 domains, notably the Region I domain, which is located close to the start site-proximal consensus promoter DNA element within the closed complex (17,16,31).…”

Section: Discussionmentioning

confidence: 99%

“…DNase I and KMnO 4 Footprinting Assays-DNase I and KMnO 4 footprinting assays on closed and open complexes (formed as described above) were conducted as described (16).…”

Section: Methodsmentioning

confidence: 99%

Stable DNA Opening within Open Promoter Complexes Is Mediated by the RNA Polymerase β′-Jaw Domain

Wigneshweraraj

Burrows

Severinov

et al. 2005

Journal of Biological Chemistry

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DNA opening for transcription-competent open promoter complex (OC) formation by the bacterial RNA polymerase (RNAP) relies upon a complex network of interactions between the structurally conserved and flexible modules of the catalytic ␤ and ␤-subunits, RNAP-associated -subunit, and the DNA. Here, we show that one such module, the ␤-jaw, functions to stabilize the OC. In OCs formed by the major 70 -RNAP, the stabilizing role of the ␤-jaw is not restricted to any particular melted DNA segment. In contrast, in OCs formed by the major variant 54 -RNAP, the ␤-jaw and a conserved 54 regulatory domain co-operate to stabilize the melted DNA segment immediately upstream of the transcription start site. Clearly, regulated communication between the mobile modules of the RNAP and the functional domain(s) of the subunit is required for stable DNA opening.The DNA-dependent RNA polymerase (RNAP) 3 is central to all steps of transcription, the most regulated stage of gene expression. The core enzymes of multisubunit RNAPs from bacteria, archaea, and eukaryotes display striking structural similarity to each other. For promoter-specific and regulated transcription to occur, the RNAP core needs to associate with auxiliary transcription initiation factors. In bacteria, RNAP core (subunit composition: ␣ 2 ␤␤Ј, E) associates with a sigma () subunit to form an RNAP holoenzyme (subunit composition: ␣ 2 ␤␤Ј, E), which is capable of promoter-specific transcription initiation. The major type of bacterial RNAP holoenzyme contains a subunit belonging to the 70 -class. In both types of bacterial RNAPs, transcription-competent open complex formation is accompanied by several conformational changes in the subunit and structurally conserved mobile modules of the core RNAP (␤-lobes, ␤-flap, ␤Ј-clamp, and the ␤Ј-jaw) (3-6). These conformational changes trigger DNA strand separation and/or accommodate the separated strands of the promoter DNA within the catalytic cleft of the RNAP. In a model of the transcription elongation complex formed by the Escherichia coli RNAP, the double-stranded DNA downstream of the catalytic center is positioned in a trough formed by the ␤Ј-jaw and the ␤Ј-clamp modules (7). The E. coli RNAP containing a deletion in the ␤Ј-jaw (⌬1149 -1190; Fig. 1A) forms unstable E 70 open complexes, although the property is evident only on some 70 -dependent promoters (8). The ␤Ј-jaw is required for several other transcription-related activities including transcriptional pausing, replication of ColEI-type plasmids, and bacteriophage M13 minus-strand DNA synthesis (8, 9). The ␤Ј-jaw is also bound by the T7 bacteriophage gp2 protein, a strong inhibitor of 70 -dependent transcription from most promoters (10, 11). The bacterial ␤Ј-jaw is the structural homologue of the RPB1 jawlobe of the eukaryotic RNAPII and its role in transcription has not been clearly determined in either RNAPs. In the bacterial RNAP, the role of the ␤Ј-jaw in binding, closed complex formation, and in steps leading to open complex formation have not been established...

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“…This helix is primarily positively charged 54 C domain (residues Ala-335 to Gly-389). Side chains for residues that were previously shown to be near the Ϫ24 promoter element (50) and/or to decrease significantly DNA binding when mutated (48) are represented as sticks and labeled (except for Tyr-384, which is hidden behind Lys-383). All of these residues are on helix 3 (dark blue) created by the highly conserved RpoN box, except for Met-343 (Arg-421 in E. coli), which is on helix 1.…”

Section: Resultsmentioning

confidence: 99%

“…3 of 70 proteins interacts with the extended Ϫ10 promoter element near the site of melting (42,49), whereas the major role of 4 of 70 proteins is to bind to the Ϫ35 promoter element (13,15). By tethering a DNA cleavage reagent, FeBABE (p-bromoacetamidobenzyl-EDTA-Fe), to charged residues in the RpoN box of K. pneumoniae 54 , Burrows et al (50) showed that residues in the RpoN box are near the Ϫ24 promoter element, not the Ϫ12 promoter element where melting occurs. For example, with the FeBABE tethered to residue E463C (A. aeolicus Glu-386), strong cleavage was seen between the Ϫ30 and Ϫ25 nucleotides of the template DNA strand.…”

Section: Discussionmentioning

confidence: 99%

The C-terminal RpoN Domain of σ54 Forms an Unpredicted Helix-Turn-Helix Motif Similar to Domains of σ70

Doucleff

Malak

Wemmer

2005

Journal of Biological Chemistry

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The "" subunit of prokaryotic RNA polymerase allows genespecific transcription initiation. Two families have been identified, 70 and 54 , which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the 70 -type factors have been well characterized structurally by x-ray crystallography, no high resolution structural information is available for the 54 -type factors. Here we present the NMR-derived structure of the C-terminal domain of 54 from Aquifex aeolicus. This domain (Thr-323 to Gly-389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the 70 -type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. The homology of this structure with other DNA-binding proteins, combined with previous biochemical data, suggests how the C-terminal domain of 54 binds to DNA.Transcription, the synthesis of RNA from double-stranded DNA, is a fundamental process in all forms of life. The primary protein complex catalyzing transcription is composed of five subunits, ␣ 2 ␤Ј␤, called "core RNA polymerase" (RNAP).2 Core RNAP is fully competent to synthesize RNA from DNA. However, the initiation of transcription at specific DNA sequences requires additional protein(s). In bacteria, these additional proteins are called the " factors" (1, 2). The factor facilitates transcription at specific DNA sequences by 1) binding to the core RNAP to form the -RNAP holoenzyme, 2) recognizing and binding to a specific DNA sequence next to the transcription start site, called the promoter element, and 3) opening the double-stranded DNA to initiate transcription.Based on protein sequence homology, factors can be grouped into two classes, 70 and 54 , which were named by the molecular weights of the first members identified (reviewed in Ref. 1 is involved in regulating the kinetics of transcription initiation; 2 binds to the Ϫ10 promoter element and is essential for melting the DNA;3 stabilizes the open complex formation by binding the extended Ϫ10 element; and 4 binds specifically to the Ϫ35 promoter element (reviewed in Ref. 17).In contrast to 70 , 54 is divided into three regions based on function (18 -20). Region 1 (E. coli-(1-55)) interacts with the upstream activator protein to control promoter melting; region 2 (E. coli-(70 -304)) contains the minimal RNAP-binding domain (E. coli-(70 -180) and a part that enhances DNA binding affinity (E. coli-(180 -304)); region 3 (E. coli-(329 -477)) recognizes and binds the consensus promoter elements. This C-terminal part of the protein contains a region that crosslinks to DNA (E. coli-(329 -346)), a predicted helix-turn-helix motif (E. coli-(366 -386); Fig. 1), and a highly conserved sequence (E. coli-* This work was supported by National Institutes of Health Grant GM62163 (to D. E. W.) and by a University of Calif...

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Mapping σ54-RNA Polymerase Interactions at the –24 Consensus Promoter Element

Cited by 30 publications

References 56 publications

Evidence that RpoS (σ ^S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ ⁵⁴ /σ ^N )

Evidence that RpoS (σ ^S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ ⁵⁴ /σ ^N )

Stable DNA Opening within Open Promoter Complexes Is Mediated by the RNA Polymerase β′-Jaw Domain

The C-terminal RpoN Domain of σ54 Forms an Unpredicted Helix-Turn-Helix Motif Similar to Domains of σ70

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Mapping σ54-RNA Polymerase Interactions at the –24 Consensus Promoter Element

Cited by 30 publications

References 56 publications

Evidence that RpoS (σ S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ 54 /σ N )

Evidence that RpoS (σ S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ 54 /σ N )

Stable DNA Opening within Open Promoter Complexes Is Mediated by the RNA Polymerase β′-Jaw Domain

The C-terminal RpoN Domain of σ54 Forms an Unpredicted Helix-Turn-Helix Motif Similar to Domains of σ70

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Evidence that RpoS (σ ^S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ ⁵⁴ /σ ^N )

Evidence that RpoS (σ ^S ) in Borrelia burgdorferi Is Controlled Directly by RpoN (σ ⁵⁴ /σ ^N )