On the Role of the Escherichia coli RNA Polymerase σ70 Region 4.2 and α-Subunit C-terminal Domains in Promoter Complex Formation on the Extended –10 galP1 Promoter

Minakhin, Leonid; Severinov, Konstantin

doi:10.1074/jbc.m304906200

Cited by 43 publications

(45 citation statements)

References 29 publications

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“…On the TS, we observed the appearance of protection at Ϫ56 to Ϫ53, Ϫ46 to Ϫ42, and Ϫ33 to Ϫ31 within the first second of the reaction. This result supports recent evidence of a specific interaction between ␣-CTD and region 4.2 of in the stabilization of initial binding (37)(38)(39). Here, we directly establish that the interaction of these two subunits with their respective binding sites occurs at the initial steps of promoter recognition.…”

Section: Structural Description Of Real-time Intermediates On the Patsupporting

confidence: 77%

Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter

Sclavi

Zaychikov

Rogozina

et al. 2005

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

125

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We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase on the T7A1 promoter at 37°C. Three sets of intermediates, corresponding to two major conformational changes, are resolved as a function of time; multiple conformations equilibrate amongst each other before the next large structural change. Analysis of these data in the context of published structural models indicates that initial recognition involves interaction of the UP element with the ␣-subunit Cterminal domain and binding of the subunit to the ؊35 sequence. In the subsequent isomerization step, two complexes with footprints extending into the ؊10 region can be differentiated as the DNA becomes distorted during nucleation of strand separation. During the final isomerization step, the downstream double helix becomes embedded in the ␤͞␤ jaws, leading to a transcriptionally active complex.DNA-protein interactions ͉ time-resolved footprinting ͉ transcription ͉ hydroxyl radicals D e novo RNA synthesis during DNA transcription is a highly regulated cellular process carried out by RNA polymerase. RNA polymerase binds to DNA and diffuses one-dimensionally to the promoter region (1-4); it then must make a number of interactions within the promoter as a prerequisite to forming a relatively stable complex. This complex is then in a state that favors isomerization, leading to strand separation from approximately Ϫ12 to ϩ3 with respect to the site of transcription initiation (5). Each of these steps is a possible site for regulation of the transcription levels. Extensive studies have been conducted on the structure of RNA polymerase-DNA complexes at equilibrium resulting in the characterization of the interaction of each of the enzyme's subunits with specific promoter elements (6, 7). However a direct characterization of the interactions formed at each step of the recognition process is still lacking.The T7A1 promoter used for the studies described here is one of the strongest known prokaryotic promoters (8). Its Ϫ35 sequence, TTGACT, is very close to the consensus (TTGACA), and its Ϫ10 sequence, GATACT, deviates from consensus (TATAAT). In addition, this promoter contains, from Ϫ42 to Ϫ80, a sequence rich in adenine and thymine residues, also known as an UP element (9-11). On other promoters this sequence has been shown to both increase the rate of promoter binding and stimulate isomerization to the open complex (12)(13)(14)(15). Kinetic studies on the formation of an RNA polymerasepromoter complex revealed the presence of a sequence of isomerization steps leading to the formation of an open complex (16-18). Furthermore, by decreasing the isomerization rates at lower temperatures, one or more intermediates in the pathway from the initial closed complex to the final open complex were isolated and characterized (19-22). However, the large, and sometimes nonlinear, temperature dependence of some ...

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Section: Structural Description Of Real-time Intermediates On the Patsupporting

confidence: 77%

Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter

Sclavi

Zaychikov

Rogozina

et al. 2005

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

125

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“…1 l of 20 mM KMnO 4 was added, and the reaction was stopped after 15 s by addition of 5 l of stop-solution containing 1 M ␤-mercaptoethanol and 1 M sodium acetate, pH 4.8. DNA was processed as described (15) and analyzed on 10% denaturing polyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

Region 3.2 of the σ Subunit Contributes to the Binding of the 3′-Initiating Nucleotide in the RNA Polymerase Active Center and Facilitates Promoter Clearance during Initiation

Kulbachinskiy

Mustaev²

2006

Journal of Biological Chemistry

130

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Region 3.2 of the RNA polymerase subunit forms a loop that protrudes toward RNA polymerase active center and partially blocks RNA exit channel. To provide some insights into the functional role of this region, we studied a deletion variant of the Escherichia coli 70 subunit that lacked amino acids 513-519 corresponding to the tip of the loop. The deletion had multiple effects on transcription initiation including: (i) a significant decrease in the amount of short abortive RNAs synthesized during initiation, (ii) defects in promoter escape, (iii) loss of the contacts between the subunit and the nascent RNA during initiation and, finally, (iv) dramatic increase in the K m value for the 3-initiating nucleotide. At the same time, the mutation did not impair promoter opening and the binding of the 5-initiating purine nucleotide. In summary, our data demonstrate an important role of region 3.2 in the binding of initiating substrates in RNA polymerase active center and in the process of promoter clearance.The subunit of bacterial RNA polymerase (RNAP) 2 plays the key role in promoter-specific initiation of RNA synthesis. The subunit is involved in multiple processes during initiation including initial promoter recognition, DNA melting, abortive synthesis, and promoter clearance (1). Recent progress in structural studies of RNAP together with the bulk of biochemical data allowed the creation of an integral picture of transcription initiation and a proposal of functional roles for individual domains of the subunit. In RNAP holoenzyme, the subunit occupies the upstream part of the main RNAP channel. Two DNA binding domains of , involved in the recognition of the Ϫ10 and Ϫ35 promoter elements, are formed by conserved regions 2 and 4 and interact with a coiled-coil element of the ␤Ј subunit and the flap domain of the ␤ subunit, respectively (2-5). The promoter recognition domains of are connected by a flexible linker formed by conserved region 3.2 (amino acids 498 -526 in Escherichia coli numbering). A hairpin-like loop from region 3.2 protrudes toward the active center of RNAP and occupies a part of the RNA exit channel (Fig. 1). This led to several predictions about the functional role of this region. First, the region 3.2 loop was proposed to directly participate in the binding of the 5Ј-initiating nucleotide in the i-site of the RNAP active center (6, 7). In support of this, E. coli 70 was shown to cross-link to a 5Ј-initiating ATP analogue in a segment between amino acids 508 and 561 containing regions 3.2 and 4.1 (8). However, no experiments were done demonstrating the functional role for this region in substrate binding. Second, region 3.2 was proposed to play an important role in the process of promoter escape (3,6,7,9). As this region blocks the path for RNA exit (Fig. 1), the elongating RNA transcript must either displace it from the RNA exit channel thus promoting dissociation or dissociate itself from the complex and be released as an abortive product. Thus, direct competition between the elongating RNA transc...

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“…To test this, we analyzed transcription by the E. coli 70 RNAP holoenzyme from two variants of the sTap2 promoter containing consensus Ϫ35 promoter element introduced 18 nucleotides upstream of the Ϫ10 element (8). Previously, we demonstrated that the Ϫ35 element artificially placed at this promoter position in galP1 and sTap2 templates could be efficiently recognized by E. coli RNAP (7,8). The first variant, sTap2ϩ35, contained both the GGGA element and the Ϫ35 element; the second, sTap2ϩ35-GGGA, contained the Ϫ35 element but had the GGGA sequence replaced with CCCT (Fig.…”

Section: E Coli 70 Region 12 Specifically Recognizes the Ggga Elemementioning

confidence: 99%

“…The T7 A1 and galP1 promoter templates were described previously (7,18). Transcription was performed in a buffer containing 40 mM HEPES, pH 7.9, 40 mM KCl, and 10 mM MgCl 2 .…”

Section: And T Aquaticusmentioning

confidence: 99%

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Structural Modules of RNA Polymerase Required for Transcription from Promoters Containing Downstream Basal Promoter Element GGGA

Barinova

Kuznedelov

Severinov

et al. 2008

Journal of Biological Chemistry

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We recently described a novel basal bacterial promoter element that is located downstream of the ؊10 consensus promoter element and is recognized by region 1.2 of the subunit of RNA polymerase (RNAP). In the case of Thermus aquaticus RNAP, this element has a consensus sequence GGGA and allows transcription initiation in the absence of the ؊35 element. In contrast, the Escherichia coli RNAP is unable to initiate transcription from GGGA-containing promoters that lack the ؊35 element. In the present study, we demonstrate that subunits from both E. coli and T. aquaticus specifically recognize the GGGA element and that the observed species specificity of recognition of GGGA-containing promoters is determined by the RNAP core enzyme. We further demonstrate that transcription initiation by T. aquaticus RNAP on GGGA-containing promoters in the absence of the ؊35 element requires region 4 and C-terminal domains of the ␣ subunits, which interact with upstream promoter DNA. When in the context of promoters containing the ؊35 element, the GGGA element is recognized by holoenzyme RNAPs from both E. coli and T. aquaticus and increases stability of promoter complexes formed on these promoters. Thus, GGGA is a bona fide basal promoter element that can function in various bacteria and, depending on the properties of the RNAP core enzyme and the presence of additional promoter elements, determine species-specific differences in promoter recognition.In bacteria, recognition of promoters is accomplished through specific interactions of the RNA polymerase (RNAP) 3 holoenzyme with consensus elements of promoter DNA. Most housekeeping promoters are recognized by RNAP holoenzyme containing the major subunit (called 70 in Escherichia coli or A in other bacteria). The Ϫ10 (TATAAT) and the Ϫ35 (TTGACA) consensus elements of these promoters interact with conserved regions 2.4 and 4.2 of , respectively (1). A subset of promoters (the so-called extended Ϫ10 promoters) contain a TG motif one nucleotide upstream of the Ϫ10 element that is recognized by region 2.5 of ; these promoters do not require the Ϫ35 element for their activity (2). Some strong promoters contain an A/T-rich UP element that is located upstream of the Ϫ35 element and is specifically recognized by the C-terminal domains of the RNAP ␣ subunits (␣CTDs) (3).In addition to specific interactions with conserved promoter elements, nonspecific RNAP-DNA interactions also contribute to promoter recognition. In particular, it was shown that nonspecific contacts of E. coli RNAP ␣CTDs with upstream DNA play an important role in promoter recognition even in the absence of the UP element (4 -6) and that contacts of 70 region 4 with DNA stimulate recognition of extended Ϫ10 promoters lacking the Ϫ35 element (7).Sigma subunits of the 70 class are unable to recognize promoters in the absence of the RNAP core. We have recently demonstrated that isolated A from thermophilic bacterium Thermus aquaticus can recognize single-stranded DNA aptamers (sTaps, for sigma T. aquaticus aptamers) that cont...

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On the Role of the Escherichia coli RNA Polymerase σ70 Region 4.2 and α-Subunit C-terminal Domains in Promoter Complex Formation on the Extended –10 galP1 Promoter

Cited by 43 publications

References 29 publications

Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter

Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter

Region 3.2 of the σ Subunit Contributes to the Binding of the 3′-Initiating Nucleotide in the RNA Polymerase Active Center and Facilitates Promoter Clearance during Initiation

Structural Modules of RNA Polymerase Required for Transcription from Promoters Containing Downstream Basal Promoter Element GGGA

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