Inhibition of Escherichia coli RNA Polymerase by Bacteriophage T7 Gene 2 Protein

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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...

Section: Discussionmentioning

confidence: 63%

mentioning

confidence: 99%

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

Wigneshweraraj

Burrows

Severinov

et al. 2005

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“…T7 phage gp2 and T4 phage AsiA proteins were prepared as described previously (27,28). E. coli core RNAP containing a deletion (1149 -1190) in the ␤Ј subunit was a generous gift of Dr. Wigneshweraraj (Imperial College, London).…”

Section: Methodsmentioning

confidence: 99%

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

Mekler

Minakhin

Severinov

2011

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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 ...

“…The samples were subjected to electrophoresis at room temperature for 2 h on a 6% polyacrylamide gel. The formation of closed complexes was determined as described by Nechaev and Severinov (22). Briefly, binding reactions with MarA (200 -1000 nM) and the rob promoter fragment (pRobF4) were performed at 0°C for 10 min prior to the addition of RNAP (40 nM).…”

mentioning

confidence: 99%

MarA-mediated Transcriptional Repression of the rob Promoter

Schneiders

Levy

2006

The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the ؊10 and ؊35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.The Rob protein is an abundant nucleoid protein that was initially discovered bound to the right origin of replication of the Escherichia coli chromosome (1). Despite its association with the origin of replication, no experimental evidence supports the role of Rob in chromosome replication, chromatin structure, and superhelicity (2).Rob is, however, a member of a subgroup within the AraC/XylS family of transcriptional regulators, which includes the MarA and SoxS proteins that are involved in a wide range of regulatory functions (3). The N-terminal domain of Rob shares similarity with MarA and SoxS, which is in contrast to the other members of the subgroup that share homology within the C-terminal domain (3). When induced by bile salts and dipyridyl, the C-terminal domain undergoes post-transcriptional modification that results in the conversion of Rob from a low to a high activity state in the cell (4). However, in the absence of these compounds, overexpression of either the full or C-terminal domain deleted protein is sufficient for activity and promoter binding in vitro (5, 6) and in vivo (5, 7). This observation is consistent with structural data derived from the Rob-micF complex, where only the N-terminal domain makes DNA-specific contacts (8). Overexpression of rob confers multidrug, organic solvent and heavy metal resistance (6, 7); in accord, some experimental data indicate that rob null mutants are hypersensitive to antibiotics, organic solvents, and heavy metals (5, 7, 9).Transposon mutagenesis experiments aimed at defining the Rob regulon revealed eight namely inaA, marRAB, aslB, ybaO, mdlA, yfhD, ybiS, and galT (10), some of which are also known members of the mar and sox regulons (11). In addition, studies demonstrate that Rob induced by decanoate or bile salts results in the increased expression of acrAB in the absence of both the mar and sox loci (12).The expression levels of rob do not vary dramatically during differ...