A Basal Promoter Element Recognized by Free RNA Polymerase σ Subunit Determines Promoter Recognition by RNA Polymerase Holoenzyme

Feklístov, Andrey; Barinova, Nataliya; Sevostyanova, Anastasiya; Heyduk, Ewa; Bass, I. A.; Vvedenskaya, Irina O.; Kuznedelov, Konstantin; Merkienė, Eglė; Stavrovskaya, Elena; Klimašauskas, Saulius; Nikiforov, Vadim; Heyduk, Tomasz; Severinov, Konstantin; Kulbachinskiy, Andrey

doi:10.1016/j.molcel.2006.06.010

Cited by 92 publications

(122 citation statements)

References 46 publications

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“…Thus, the GGGA motif is a bona fide basal promoter element for T. aquaticus RNAP. Indeed, we found that similar motifs are present in natural promoters and increase stability of promoter complexes of RNAPs from T. aquaticus and related bacteria (8). Bioinformatics analysis suggested that up to 33% of predicted promoters in the genome of Thermus thermophilus contain GGGA-like motifs.…”

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confidence: 85%

“…Bioinformatics analysis suggested that up to 33% of predicted promoters in the genome of Thermus thermophilus contain GGGA-like motifs. In contrast, no preference for such motifs was found in E. coli promoter sequences (8).…”

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confidence: 86%

“…Surprisingly, however, RNAPs from T. aquaticus and E. coli were found to differ in recognition of promoters containing the GGGA element (8). Although in the case of T. aquaticus RNAP, the Ϫ10 element and the GGGA element were sufficient for transcription initiation, E. coli RNAP required the Ϫ35 element whether the GGGA element was present or not.…”

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confidence: 96%

“…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 contain a Ϫ10-like element followed by the tetranucleotide motif GGGA (8). Using site-specific DNA-protein cross-linking, we demonstrated that the GGGA motif interacts with region 1.2 of A .…”

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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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confidence: 85%

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confidence: 86%

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confidence: 96%

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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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“…1): the UP element (bound by the C-terminal domains of the two ␣ subunits); the Ϫ35 hexamer (bound by region 4.2); the extended Ϫ10 element (bound by region 3.0); and the Ϫ10 hexamer (bound by region 2.3-2.4). Recently, an additional element immediately downstream of the Ϫ10 hexamer, within the discriminator region, was proposed to bind to region 1.2 (3,4).…”

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confidence: 99%

Fine structure of the promoter–σ region 1.2 interaction

Haugen

Ross

Manrique

et al. 2008

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

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We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the ؊10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with 1.2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases at the junction of the ؊10 element and the discriminator region in an unregulated rRNA promoter variant and in the PR promoter. Our studies indicate that nontemplate strand bases adjacent to the ؊10 element bind within a 9-aa interval in 1.2 (residues 99 -107). We also demonstrate that the downstream-most base on the nontemplate strand of the ؊10 hexamer cross-links to region 2, and not to 1.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation.promoter element ͉ RNA polymerase ͉ transcription initiation ͉ discriminator region ͉ Ϫ10 element I nteractions between bacterial RNA polymerase holoenzyme (RNAP; ␣ 2 ␤␤Ј ) and the promoter can determine not only its basal strength but also its regulation. (In this report, always refers to 70 , the major factor.) Four promoter elements are generally recognized as making sequence-specific contacts with RNAP (1, 2) ( Fig. 1): the UP element (bound by the C-terminal domains of the two ␣ subunits); the Ϫ35 hexamer (bound by region 4.2); the extended Ϫ10 element (bound by region 3.0); and the Ϫ10 hexamer (bound by region 2.3-2.4). Recently, an additional element immediately downstream of the Ϫ10 hexamer, within the discriminator region, was proposed to bind to region 1.2 (3, 4).The term ''discriminator'' was coined by Travers (5) more than 25 years ago to describe a GϩC-rich region downstream from the Ϫ10 hexamer in stable RNA (rRNA and tRNA) promoters, and it was proposed that the GϩC content of this region was important for maintaining proper regulation of stable RNA promoters (6, 7). High GϩC content was proposed to impede strand separation, leading to promoter regulation. However, it was found that a C to G substitution 2 nt downstream from the Ϫ10 hexamer in the rRNA promoter rrnB P1 (rrnB P1 C-7G) eliminated its regulation, suggesting that the actual sequence of the discriminator region, in addition to its high GϩC content, is crucial for control of transcription (3).Footprinting, photocross-linking, and genetic approaches led to the conclusion that the nontemplate strand base two positions downstream from the Ϫ10 element in the rrnB P1 C-7G promoter contacts 1.2. When the base at the analogous position in all other promoters investigated was a C (either naturally or by mutation), competitor-resistant complexes formed with RNAP were much shorte...

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Use of RNA Polymerase Molecular Beacon Assay to Measure RNA Polymerase Interactions with Model Promoter Fragments

Mekler

Severinov

2015

Methods in Molecular Biology

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RNA polymerase-promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase-promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase-promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase-promoter interactions. Here, we present detailed protocols for the preparation of an Escherichia coli molecular beacon and its application to study RNA polymerase interactions with model promoter fragments.

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A Basal Promoter Element Recognized by Free RNA Polymerase σ Subunit Determines Promoter Recognition by RNA Polymerase Holoenzyme

Cited by 92 publications

References 46 publications

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

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

Fine structure of the promoter–σ region 1.2 interaction

Use of RNA Polymerase Molecular Beacon Assay to Measure RNA Polymerase Interactions with Model Promoter Fragments

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