scholarly journals Characterization of PmfR, the Transcriptional Activator of the pAO1-Borne purU-mabO-folD Operon of Arthrobacter nicotinovorans

2005 ◽  
Vol 187 (9) ◽  
pp. 3062-3070 ◽  
Author(s):  
Calin B. Chiribau ◽  
Cristinel Sandu ◽  
Gabor L. Igloi ◽  
Roderich Brandsch
Keyword(s):  
Binding Site ◽  
Transcriptional Start Site ◽  
Transcriptional Activator ◽  
Open Reading Frames ◽  
Start Site ◽  
Gene Encoding ◽  
Chloramphenicol Resistance ◽  
Nuclease Digestion ◽  
Arthrobacter Nicotinovorans

ABSTRACT Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation γ-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating γ-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between −48 and −88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the −35 promoter region of the purU-mabO-folD operon.

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

1991 ◽  
Vol 11 (2) ◽  
pp. 1048-1061
Author(s):  
I J Lee ◽  
L Tung ◽  
D A Bumcrot ◽  
E S Weinberg
Keyword(s):  
Binding Site ◽  
Sea Urchin ◽  
Transcriptional Start Site ◽  
Sodium Dodecyl ◽  
Nuclear Extract ◽  
Dnase I ◽  
Histone H4 ◽  
Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes

2000 ◽  
Vol 3 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Q. XIE ◽  
D. H. ALPERS
Keyword(s):  
Alkaline Phosphatase ◽  
Transcriptional Start Site ◽  
In Vivo Studies ◽  
Peroxisome Proliferator ◽  
Start Site ◽  
Intestinal Alkaline Phosphatase ◽  
Promoter Regions ◽  
Flanking Regions

Xie, Q., and D. H. Alpers. The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes. Physiol Genomics 3: 1–8, 2000.—Rat intestinal alkaline phosphatases (IAP-I and -II) differ in primary structure, substrate specificity, tissue localization, and response to fat feeding. This study identifies two distinct genes (∼5–6 kb) corresponding to each isozyme and containing 11 exons of nearly identical size. The exon-intron junctions are identical with those found in IAP genes from other species. The 1.7 and 1.2 bp of 5′ flanking regions isolated from each gene, respectively, contain Sp1 and gut-enriched Kruppel-like factor (GKLF) binding sites, but otherwise show little identity. There is a potential CAAT-box 14 bp 5′ to the transcriptional start site, 36 bp upstream from IAP-I, and a TATA-box 31 bp 5′ to the transcriptional start site, 55 bp upstream from IAP-II. Transfection of these promoter regions (linked to luciferase as a reporter gene) into a kidney cell line, COS-7, produced the differential response to oleic acid expected from in vivo studies, i.e., threefold increase using the 5′ flanking region of IAP-II, but not IAP-I. This response was not reproduced by 5,8,11,14-eicosatetraynoic acid (ETYA) or clofibrate, suggesting that peroxisome proliferator response elements are not involved. Isolation of the IAP-II gene will allow determination of the sequences responsible for dietary fat response in the enterocyte.

scholarly journals Binding Site Determinants for the LysR-Type Transcriptional Regulator PcaQ in the Legume Endosymbiont Sinorhizobium meliloti

2007 ◽  
Vol 190 (4) ◽  
pp. 1237-1246 ◽  
Author(s):  
Allyson M. MacLean ◽  
Michelle I. Anstey ◽  
Turlough M. Finan
Keyword(s):  
Binding Site ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Transcriptional Start Site ◽  
Mesorhizobium Loti ◽  
Equilibrium Dissociation ◽  
Dyad Symmetry

ABSTRACT LysR-type transcriptional regulators represent one of the largest groups of prokaryotic regulators described to date. In the gram-negative legume endosymbiont Sinorhizobium meliloti, enzymes involved in the protocatechuate branch of the β-ketoadipate pathway are encoded within the pcaDCHGB operon, which is subject to regulation by the LysR-type protein PcaQ. In this work, purified PcaQ was shown to bind strongly (equilibrium dissociation constant, 0.54 nM) to a region at positions −78 to −45 upstream of the pcaD transcriptional start site. Within this region, we defined a PcaQ binding site with dyad symmetry that is required for regulation of pcaD expression in vivo and for binding of PcaQ in vitro. We also demonstrated that PcaQ participates in negative autoregulation by monitoring expression of pcaQ via a transcriptional fusion to lacZ. Although pcaQ homologues are present in many α-proteobacteria, this work describes the first reported purification of this regulator, as well as characterization of its binding site, which is conserved in Agrobacterium tumefaciens, Rhizobium leguminosarum, Rhizobium etli, and Mesorhizobium loti.

scholarly journals Joint Transcriptional Control of xpsR, the Unusual Signal Integrator of the Ralstonia solanacearum Virulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator

1998 ◽  
Vol 180 (10) ◽  
pp. 2736-2743 ◽  
Author(s):  
Jianzhong Huang ◽  
Wandee Yindeeyoungyeon ◽  
Ram P. Garg ◽  
Timothy P. Denny ◽  
Mark A. Schell
Keyword(s):  
Binding Site ◽  
Transcriptional Control ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Virulence Gene ◽  
Transcriptional Activator ◽  
Binding Assays ◽  
Environmental Signals

ABSTRACT Ralstonia (Pseudomonas)solanacearum is a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of the epsoperon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that thexpsR promoter (P xpsR ) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into theeps promoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of P xpsR to show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position −77, causing a sixfold activation of P xpsR ; a weaker, less-defined binding site between −183 and −239 likely enhances PhcA binding and activation via the −77 site another twofold. (ii) Full 70-fold activation of P xpsR requires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around −315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate P xpsR . (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect P xpsR activation or regulation.

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 NtcA is responsible for accumulation of the small isoform of ferredoxin:NADP oxidoreductase

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 789-794 ◽  
Author(s):  
Amin Omairi-Nasser ◽  
Carla V. Galmozzi ◽  
Amel Latifi ◽  
M. Isabel Muro-Pastor ◽  
Ghada Ajlani
Keyword(s):  
Binding Site ◽  
Translation Initiation ◽  
Transcriptional Regulator ◽  
Growth Conditions ◽  
Gene Encoding

In several cyanobacteria, petH, the gene encoding ferredoxin:NADP oxidoreductase (FNR), is transcribed from at least two promoters depending on growth conditions. Two transcripts (short and long) are translated from two different translation initiation sites, resulting in two isoforms (large and small, respectively). Here, we show that in Synechocystis PCC6803 the global transcriptional regulator NtcA activates transcription from the distal petH promoter. Modification of the NtcA-binding site prevents NtcA binding to the promoter in vitro and abolishes accumulation of the small isoform of FNR in vivo. We also demonstrate that a similar petH transcription and translation regime occurs in other cyanobacteria. The conditions under which this system operates provide hints for the function of each FNR isoform.

scholarly journals Transcriptional Regulation ofFucosyltransferase 1Gene Expression in Colon Cancer Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Fumiko Taniuchi ◽  
Koji Higai ◽  
Tomomi Tanaka ◽  
Yutaro Azuma ◽  
Kojiro Matsumoto
Keyword(s):  
Gene Expression ◽  
Colon Cancer ◽  
Transcriptional Regulation ◽  
Binding Site ◽  
Transcriptional Start Site ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Start Site ◽  
Lewis Y

Theα1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses theFUT1gene, and showsα1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay,FUT1gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation ofFUT1gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression ofFUT1is regulated by Elk-1 in DLD-1 cells.

scholarly journals The Novel Transcription Factor SgrR Coordinates the Response to Glucose-Phosphate Stress

2007 ◽  
Vol 189 (6) ◽  
pp. 2238-2248 ◽  
Author(s):  
Carin K. Vanderpool ◽  
Susan Gottesman
Keyword(s):  
Binding Site ◽  
Binding Domain ◽  
Alternative Mechanism ◽  
Gene Encoding ◽  
Bacterial Transcription ◽  
Glucose Phosphate ◽  
Plasmid Library ◽  
Promoter Dna

ABSTRACT SgrR is the first characterized member of a family of bacterial transcription factors containing an N-terminal DNA binding domain and a C-terminal solute binding domain. Previously, we reported genetic evidence that SgrR activates the divergently transcribed gene sgrS, which encodes a small RNA required for recovery from glucose-phosphate stress. In this study, we examined the regulation of sgrR expression and found that SgrR negatively autoregulates its own transcription in the presence and absence of stress. An SgrR binding site in the sgrR-sgrS intergenic region is required in vivo for both SgrR-dependent activation of sgrS and autorepression of sgrR. Purified SgrR binds specifically to sgrS promoter DNA in vitro; a mutation in the site required for in vivo activation and autorepression abrogates in vitro SgrR binding. A plasmid library screen identified clones that alter expression of a P sgrS -lacZ fusion; some act by titrating endogenous SgrR. The yfdZ gene, encoding a putative aminotransferase, was identified in this screen; the yfdZ promoter contains an SgrR binding site, and transcriptional fusions indicate that yfdZ is activated by SgrR. Clones containing mlc, which encodes a glucose-specific repressor protein, also downregulate P sgrS -lacZ. The mlc clones do not appear to titrate the SgrR protein, indicating that Mlc affects sgrS expression by an alternative mechanism.

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