scholarly journals Mechanism of Repression of the aroP P2 Promoter by the TyrR Protein of Escherichia coli

1999 ◽  
Vol 181 (20) ◽  
pp. 6411-6418 ◽  
Author(s):  
Ji Yang ◽  
Peixiang Wang ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Binding Site ◽  
Mutational Analysis ◽  
Critical Role ◽  
Start Site ◽  
Transcription Start ◽  
P2 Promoter

ABSTRACT Previously, we have shown that expression of the Escherichia coli aroP P2 promoter is partially repressed by the TyrR protein alone and strongly repressed by the TyrR protein in the presence of the coeffector tyrosine or phenylalanine (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Here we present in vitro results showing that the TyrR protein and RNA polymerase can bind simultaneously to the aroP P2 promoter. In the presence of tyrosine, the TyrR protein inhibits open complex formation at the P2 promoter, whereas in the absence of any coeffector or in the presence of phenylalanine, the TyrR protein inhibits a step(s) following the formation of open complexes. We also present mutational evidence which implicates the N-terminal domain of the TyrR protein in the repression of P2 expression. The TyrR binding site of aroP, which includes one weak and one strong TyrR box, is located 5 bp downstream of the transcription start site of P2. Results from a mutational analysis show that the strong box (which is located more closely to the P2 promoter), but not the weak box, plays a critical role in P2 repression.

scholarly journals Activation of Escherichia coli leuVTranscription by FIS

1999 ◽  
Vol 181 (12) ◽  
pp. 3864-3868 ◽  
Author(s):  
Wilma Ross ◽  
Julia Salomon ◽  
Walter M. Holmes ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Stable Complex ◽  
Start Site ◽  
Transcription Start ◽  
Transcription In Vitro ◽  
A Site

ABSTRACT The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1 Leu. However, no evidence for direct involvement of FIS in tRNA1 Leu expression has been reported. We show here that FIS binds to a site upstream of the leuVpromoter (centered at −71) and that it directly stimulatesleuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of ∼−7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.

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 Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 5993-6004 ◽  
Author(s):  
Anne M. L. Barnard ◽  
Jeffrey Green ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Binding Site ◽  
Transcription Regulation ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Start Site ◽  
Transcription Start ◽  
Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

scholarly journals Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

2020 ◽  
Author(s):  
Zhe Sun ◽  
Alexander Yakhnin ◽  
Peter C. FitzGerald ◽  
Carl E. Mclntosh ◽  
Mikhail Kashlev
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Environmental Cues ◽  
Start Site ◽  
Transcription Start ◽  
E Coli ◽  
Eukaryotic Genes ◽  
Genome Wide ◽  
Transcript Cleavage

ABSTRACTPromoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of σ70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of σ70 to canonical −10 element, 6-7 nt spacer and “YR+1Y” motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional −10-like sequence positioned on the same face of the DNA duplex as the original −10 element suggesting that σ70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

scholarly journals Cra-Dependent Transcriptional Activation of theicd Gene of Escherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 893-898 ◽  
Author(s):  
Jean-François Prost ◽  
Didier Nègre ◽  
Christelle Oudot ◽  
Katsuhiko Murakami ◽  
Akira Ishihama ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Α Subunit ◽  
P2 Promoter ◽  
Catabolite Repressor ◽  
A Site ◽  
Extension Analysis

ABSTRACT The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position −76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the α subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

scholarly journals Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2359-2366 ◽  
Author(s):  
Ming Tan ◽  
Tamas Gaal ◽  
Richard L. Gourse ◽  
Joanne N. Engel
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Rich Region ◽  
E Coli ◽  
Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

scholarly journals The organization of open complexes between Escherichia coli RNA polymerase and DNA fragments carrying promoters either with or without consensus −35 region sequences

1990 ◽  
Vol 270 (1) ◽  
pp. 141-148 ◽  
Author(s):  
B Chan ◽  
A Spassky ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Point Mutations ◽  
Dna Fragments ◽  
Transcription Start ◽  
Footprint Analysis ◽  
Open Complex Formation ◽  
Promoter Dna

Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region. Footprint analysis of transcriptionally competent complexes between E. coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed. In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected. Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA. However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence. With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced. In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone. We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation.

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 Role of Region C in Regulation of the Heat Shock Gene-Specific Sigma Factor of Escherichia coli, ς32

1999 ◽  
Vol 181 (11) ◽  
pp. 3552-3561 ◽  
Author(s):  
Florence Arsène ◽  
Toshifumi Tomoyasu ◽  
Axel Mogk ◽  
Christiane Schirra ◽  
Agnes Schulze-Specking ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Binding Site ◽  
Heat Shock Gene ◽  
Ftsh Protease ◽  
Shock Gene ◽  
Stress Dependent

ABSTRACT Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the ς32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates ς32 by stress-dependent association and mediates ς32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among ς32 homologs, termed region C, was proposed to play a role in ς32degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of ς32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the ς32 protein did not affect ς32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in ς32 control by DnaK and FtsH. Instead, the ς32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of ς32 to RNA polymerase. Region C thus confers on ς32 a competitive advantage over other ς factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.

Sign in / Sign up

Export Citation Format

Share Document