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

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

doi:10.1074/jbc.m506416200

Cited by 21 publications

(31 citation statements)

References 35 publications

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“…In order to directly establish whether RPo formation occurs at 37°C under our experiment conditions (and also to distinguish the RPc and RPo without the use of heparin or chemical probes), we used an assay that directly reports transcriptionally proficient DNA melting and RPo formation. The assay, hereafter referred to as the short RNA synthesis assay (which we routinely use to measure RPo formation by E. coli Eσ 70 ), 28,29 measures the ability of Eσ A to synthesise a dinucleotide-primed RNA product from the P2 and P3 promoters. As shown in Fig [α-32 P]UTP (which complements position + 4 of the P3 template strand; Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular Insights into the Control of Transcription Initiation at the Staphylococcus aureus agr operon

Reynolds

Wigneshweraraj

2011

Journal of Molecular Biology

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The accessory gene regulatory (agr) operon of the opportunistic human pathogen Staphylococcus aureus is a prime pathogenesis factor in this bacterium. The agr operon consists of two transcription units, RNAII and RNAIII, which are transcribed from divergent promoters, P2 and P3, respectively. RNAII encodes a quorum-sensing system, including AgrA, the master transcription activator of the agr operon. RNAIII is the effector RNA molecule that regulates the expression of many virulence genes. Owing to the atypical spacer lengths of P2 and P3, it is widely considered that transcription from P2 and P3 only occurs in a strictly AgrA-dependent manner. Here, using a fully native S. aureus in vitro transcription system, we provide the first molecular and mechanistic characterisation of the regulation of transcription initiation at the agr operon. Surprisingly, the results demonstrate that RNA polymerase (RNAp) can interact with P2 and P3 equally well in the absence of AgrA. However, formation of a transcription-competent open promoter complex (RPo) occurs more readily at P2 than at P3 when AgrA is absent. Reducing the atypical P3 spacer region length to the optimal length of 17 nucleotides significantly improves promoter activity by facilitating the isomerisation of the initial RNAp-P3 complexes to RPo, and the extended -10-like element of P3 is required for optimal promoter activity. AgrA increases the occupancy of both promoters by RNAp and thereby increases the amount of transcription initiated at P2 and P3. However, the AgrA-mediated effect on transcription initiation is more prominent at P3 that at P2. The effect of AgrA at P2 and P3 appears to be restricted to events leading to the formation of RPo. The relevance of AgrA-independent and AgrA-dependent transcription initiation at P2 and P3 is presented in the context of our current understanding of the role of the agr operon in the pathobiology of S. aureus.

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Section: Resultsmentioning

confidence: 99%

Molecular Insights into the Control of Transcription Initiation at the Staphylococcus aureus agr operon

Reynolds

Wigneshweraraj

2011

Journal of Molecular Biology

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“…Thus, the results from the set of experiments presented here clearly point toward an involvement of 54 Region I in the sensing and communicating of information relating to regulatory center conformation by the GAFTGA motif during open complex formation. The interplay between 54 Region I and the GAFTGA motif during open complex formation is of functional significance because 54 Region I also appears to determine the activities of three structurally conserved RNAP domains (␤Ј lobe, ␤Ј jaw, and ␤Ј clamp) that contribute to a DNA-binding channel in RNAP, where DNA downstream of the RNAP active center lies, and ensure that stable DNA opening near the transcription start site is maintained (7,23). Previously, we proposed that 54 Region I acts as a "relay" domain and communicates with the downstream DNA-binding channel in RNAP in response to activation (24).…”

Section: Discussionmentioning

confidence: 99%

A Role for the Conserved GAFTGA Motif of AAA+ Transcription Activators in Sensing Promoter DNA Conformation

Dago

Wigneshweraraj

Buck

et al. 2007

Journal of Biological Chemistry

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Gene transcription in bacteria is catalyzed by the multisubunit DNA-dependent RNA polymerase (RNAP).5 The catalytically competent core form of bacterial RNAP is a five-subunit enzyme (␣ 2 ␤␤Ј; E), which has to associate with a sixth subunit, the factor, for promoter-specific and regulated initiation of gene transcription. On the basis of differences in mechanism of action and amino acid sequence, bacterial factors are classified into two families. Most bacterial factors belong to the 70 family, named after the prototypical housekeeping factor, 70 , of Escherichia coli. The major variant bacterial factor belongs to the 54 class. Transcription initiation by RNAP containing 70 (E 70 ) and 54 (E 54 ) is mechanistically distinct. E 70 recognizes promoters that contain consensus sequences centered at DNA positions Ϫ35 and Ϫ10, respectively, from the transcription start site (at ϩ1). The initial transcriptionally inactive E 70 ⅐DNA complex, called the closed complex, can spontaneously isomerize to form the transcriptionally active open complex, in which the DNA strands are separated and the RNAP is poised for RNA synthesis. In contrast, E 54 forms closed complexes on promoters that contain consensus sequences centered at DNA positions Ϫ24 and Ϫ12 (1). Closed complexes formed by E 54 remain inactive for transcription unless activated by a specialized type of transcription activator protein that belongs to the AAA (ATPases associated with various cellular activities) protein family (2). E 54 -dependent transcription activators (from now on referred to as AAA activators) bind to DNA sites located (ϳ150 -200 bases) upstream of the promoter (known as upstream activating sequences) and use the energy derived from ATP binding and hydrolysis to remodel the E 54 closed complex (2). The ATP hydrolysis-dependent binding interactions between the AAA activator and E 54 closed complex trigger a series of protein and DNA isomerization events in the E 54 closed complex, which result in the formation of the open complex. The major energetically favorable binding site for the AAA activator within the E 54 closed complex is the N-terminal Region I domain of 54 (see Fig. 1A) (3), which, in the closed complex, is located at the Ϫ12 consensus promoter region, where DNA opening for open complex formation nucleates (4). At the Ϫ12 promoter region, 54 Region I meditates tight binding to a repressive fork junction structure and so prevents open complex formation in the absence of activation. Region I of 54 is associated with a range of properties of E 54 (1). These include maintaining the closed complex transcriptionally silent prior to activation (5), stabilizing the open complex once it is formed (6), and conformational signaling to a structurally conserved DNA-interacting domain(s) of the catalytic ␤Ј subunit of RNAP (7) required for stable open complex formation. Region I of 54 has been shown to make extensive interactions with the catalytic ␤ and ␤Ј subunits of RNAP (8).The AAA activators of E 54 are mechanochemical P-loop ATPases of th...

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“…It has been previously proposed that RNAP contacts with an ϳ20-nt-long promoter DNA duplex located downstream of the transcription start can play a significant role in stabilization of RP o (16,43,44). Our direct measurements of RNAP binding to a set of downstream fork junctions reveal that RNAP affinity to the downstream duplex is quite high.…”

Section: Discussionmentioning

confidence: 99%

A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex

Mekler

Minakhin

Severinov

2011

Journal of Biological Chemistry

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Formation of the transcription-competent open promoter complex (RP o ) by DNA-dependent RNA polymerase (RNAP) 3 is a critical checkpoint on the pathway of gene expression. In bacteria, the transition from initial recognition of the promoter by RNAP to RP o proceeds through a series of short-lived intermediate complexes (1-3). In RP o , the DNA duplex is disrupted over a stretch of 12-15 base pairs, which leads to the formation of the transcription bubble and makes the transcription start point (position ϩ1) accessible to the RNAP catalytic center (4, 5). An important source of energy driving this strand separation process is the interaction of the RNAP 70 subunit region 2 with the non-template strand of the Ϫ10 promoter element at the upstream edge of the transcription bubble (6 -9). The interaction with the Ϫ10 element adenine at position Ϫ11 of the nontemplate strand is of special importance for nucleation of promoter melting (6, 10). RNAP interaction with the template segment of the transcription bubble was estimated to be considerably weaker than that with the non-template segment (8). Although formation of intermediates on the pathway to RP o has been characterized kinetically in many studies (1-3, 11, 12), it is still unclear which energetically favorable RNAP-promoter interactions are coupled with downstream propagation of the transcription bubble and determine its final boundary position at ϩ2/ϩ3.The crystal structures of prokaryotic RNAP indicate that establishment of RNAP interactions with the downstream duplex upon RP o formation must introduce a sharp kink in DNA, which may facilitate melting and stabilize the open complex (13,14). Indeed, RNAP mutations that change RNAP contacts with downstream DNA reduce the longevity of open complexes formed at certain promoters (15, 16). On highly supercoiled DNA, the downstream interactions may not be obligatory, as an RNAP subassembly containing an N-terminal fragment of the Escherichia coli RNAP ␤Ј subunit (lacking ␤Ј amino acids involved in contacts with downstream DNA) and a fragment of 70 subunit was reported to recognize and melt an extended Ϫ10 consensus promoter (17). However, melting observed in these experiments might not necessarily mirror steps in RP o formation by RNAP (3, 18). Downstream RNAPpromoter contacts are targeted by low molecular weight inhibitors and protein repressors that affect transcription initiation (16, 19 -21).Unraveling the role of downstream RNAP-promoter interactions in open complex formation and regulation of transcription initiation is hindered by the lack of experimental tools to directly measure their strength and specificity. Upon formation of the open promoter complex, two DNA fork junction structures are created around positions Ϫ12 and ϩ2. Studies of RNAP interactions with model DNA fragments mimicking the upstream fork junction provided important structural (22) and functional insights into the processes of promoter recognition and melting (8,9,(23)(24)(25)(26). Here, we introduce downstream fork junction fragments as ...

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

Cited by 21 publications

References 35 publications

Molecular Insights into the Control of Transcription Initiation at the Staphylococcus aureus agr operon

Molecular Insights into the Control of Transcription Initiation at the Staphylococcus aureus agr operon

A Role for the Conserved GAFTGA Motif of AAA+ Transcription Activators in Sensing Promoter DNA Conformation

A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex

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