DNA binding properties of the Escherichia coli nitric oxide sensor NorR: towards an understanding of the regulation of flavorubredoxin expression

2005 ◽  
Vol 33 (1) ◽  
pp. 181-183 ◽  
Author(s):  
N. Tucker ◽  
B. D'Autréaux ◽  
S. Spiro ◽  
R. Dixon
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Start Site ◽  
Binding Sites ◽  
Regulatory Protein ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Start Site ◽  
Transcription Start

Nitric oxide is an intermediate of denitrification, and is one of the radical species deployed by macrophages against invading pathogens, therefore bacterial responses to NO are of considerable importance. The Escherichia coli flavorubredoxin and its associated oxidoreductase reduce NO to nitrous oxide under anaerobic conditions, and are encoded by the norVW transcription unit. Expression of norVW requires the NO sensing regulatory protein NorR and is dependent on RNA polymerase containing the alternative sigma factor, σ54. We have purified NorR and shown that it binds to three sites in the norVW promoter region, located 75–140 bp upstream of the experimentally verified transcription start site. We have also identified two binding sites for the integration host factor, one between the NorR sites and the σ54-RNA polymerase binding site, and a second downstream of the norVW transcription start site. Comparison of the norVW promoters of enteric bacteria along with known and putative NorR-regulated promoters from Vibrio, Ralstonia and Pseudomonas species suggests that NorR binding sites contain an invariant GT(N7)AC motif flanking an AT-rich central region. The identification of a consensus for NorR binding sites will help to elucidate additional members of the NorR regulon.

scholarly journals T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site

2010 ◽  
Vol 107 (5) ◽  
pp. 2247-2252 ◽  
Author(s):  
Beatriz Cámara ◽  
Minhao Liu ◽  
Jonathan Reynolds ◽  
Andrey Shadrin ◽  
Bing Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Start Site ◽  
Structural Model ◽  
Start Site ◽  
Transcription Start ◽  
Strand Separation ◽  
Arginine Residues ◽  
Dna Strand ◽  
T7 Phage

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the host RNA polymerase (RNAP)—a multi-subunit enzyme responsible for gene transcription—by a small (∼7 kDa) phage-encoded protein called Gp2. Gp2 is also a potent inhibitor of E. coli RNAP in vitro. Here we describe the first atomic resolution structure of Gp2, which reveals a distinct run of surface-exposed negatively charged amino acid residues on one side of the molecule. Our comprehensive mutagenesis data reveal that two conserved arginine residues located on the opposite side of Gp2 are important for binding to and inhibition of RNAP. Based on a structural model of the Gp2-RNAP complex, we propose that inhibition of transcription by Gp2 involves prevention of RNAP-promoter DNA interactions required for stable DNA strand separation and maintenance of the “transcription bubble” near the transcription start site, an obligatory step in the formation of a transcriptionally competent promoter complex.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

2005 ◽  
Vol 280 (50) ◽  
pp. 41315-41323 ◽  
Author(s):  
Patricia Domínguez-Cuevas ◽  
Patricia Marín ◽  
Juan L. Ramos ◽  
Silvia Marqués
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Start Site ◽  
Transcription Start

scholarly journals Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Daniel P. Kiesenhofer ◽  
Robert L. Mach ◽  
Astrid R. Mach-Aigner
Keyword(s):  
Trichoderma Reesei ◽  
Transcription Start Site ◽  
Binding Sites ◽  
Inverted Repeat ◽  
Promoter Activity ◽  
Promoter Strength ◽  
Start Site ◽  
Transcription Start ◽  
Content Type ◽  
Sheer Number

ABSTRACTTrichoderma reeseican produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of thecbh1promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in thexyn1promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One suchciselement, an inverted repeat, plays a crucial role in the inducibility of thexyn1promoter. We investigated the impact of the properties of suchciselements by shuffling them by insertion, exchange, deletion, and rearrangement ofciselements in both thecbh1andxyn1promoter. A promoter-reporter assay using theAspergillus nigergoxAgene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase incbh1promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances ofciselements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances ofciselements have large impacts on the strength of thecbh1promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically importantcbh1promoter can be improved byciselement rearrangement.IMPORTANCEIn the present study, we demonstrate that the arrangement ofciselements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances ofciselements to the transcription start site. Furthermore, we found that the configuration ofciselements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement ofciselements is an important factor that should not be overlooked when enhancement of gene expression is desired.

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