scholarly journals Regulation of the Carnitine Pathway inEscherichia coli: Investigation of the cai-fixDivergent Promoter Region

1998 ◽  
Vol 180 (10) ◽  
pp. 2599-2608 ◽  
Author(s):  
Anne Buchet ◽  
Knut Eichler ◽  
Marie-Andrée Mandrand-Berthelot
Keyword(s):  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Start Site ◽  
Direct Role ◽  
Dnase I Footprinting ◽  
Carnitine Metabolism

ABSTRACT The divergent structural operons caiTABCDE andfixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopylacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in anfnr mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at −41.5 of the caitranscriptional start site, plays a direct role in the solecai activation. In contrast, mutation in CRP site 2, positioned at −69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at −126.5 of fix, might be to reinforce the action of site 2. A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.

scholarly journals The chromatin remodeler Ino80 mediates RNAPII pausing site determination

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Youngseo Cheon ◽  
Sungwook Han ◽  
Taemook Kim ◽  
Daehee Hwang ◽  
Daeyoup Lee
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Start Site ◽  
Regulation Of Gene Expression ◽  
Embryonic Stem ◽  
Start Site ◽  
Chromatin Remodeler ◽  
Close Relationship ◽  
Early Transcription ◽  
Different Characteristics

Abstract Background Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. Results Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. Conclusions Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

scholarly journals Compensatory periplasmic nitrate reductase activity supports anaerobic growth ofPseudomonas aeruginosaPAO1 in the absence of membrane nitrate reductase

2009 ◽  
Vol 55 (10) ◽  
pp. 1133-1144 ◽  
Author(s):  
Nadine E. Van Alst ◽  
Lani A. Sherrill ◽  
Barbara H. Iglewski ◽  
Constantine G. Haidaris
Keyword(s):  
Nitrate Reductase ◽  
Response Regulator ◽  
Reductase Activity ◽  
Transcriptional Start Site ◽  
Atp Synthesis ◽  
Anaerobic Growth ◽  
Start Site ◽  
Terminal Electron Acceptor ◽  
Periplasmic Nitrate Reductase ◽  
Growth Recovery

Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa . Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase.

scholarly journals Independent Regulation of MucD, an HtrA-Like Protease in Pseudomonas aeruginosa, and the Role of Its Proteolytic Motif in Alginate Gene Regulation

2006 ◽  
Vol 188 (8) ◽  
pp. 3134-3137 ◽  
Author(s):  
Lynn F. Wood ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Regulation ◽  
Transcriptional Start Site ◽  
Single Copy ◽  
Start Site ◽  
Temperature Resistance

ABSTRACT Expression of mucD, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in Pseudomonas aeruginosa. Expressed from the algT-mucABCD operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in algT or mucB. A transcriptional start site for a mucD promoter (PmucD) was mapped within mucC. Expression of single-copy mucD217, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.

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 Characterization of Enhancer Binding by the Vibrio cholerae Flagellar Regulatory Protein FlrC

2005 ◽  
Vol 187 (9) ◽  
pp. 3158-3170 ◽  
Author(s):  
Nidia E. Correa ◽  
Karl E. Klose
Keyword(s):  
Binding Site ◽  
Vibrio Cholerae ◽  
Transcription Start Site ◽  
Regulatory Protein ◽  
Dnase I ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Dnase I Footprinting

ABSTRACT The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence. It has previously been demonstrated that the σ54-dependent activator FlrC is necessary for both flagellar synthesis and for enhanced intestinal colonization. In order to characterize FlrC binding, we analyzed two FlrC-dependent promoters, the highly transcribed flaA promoter and the weakly transcribed flgK promoter, utilizing transcriptional lacZ fusions, mobility shift assays, and DNase I footprinting. Promoter fusion studies showed that the smallest fragment with wild-type transcriptional activity for flaAp was from positions −54 to +137 with respect to the start site, and from −63 to +144 for flgKp. Gel mobility shift assays indicated that FlrC binds to a fragment containing the region from positions +24 to +95 in the flaAp, and DNase I footprinting identified a protected region between positions +24 and +85. Mobility shift and DNase I footprinting indicated weak binding of FlrC to a region downstream of the flgKp transcription start site. These results demonstrate a relatively novel σ54-dependent promoter architecture, with the activator FlrC binding downstream of the σ54-dependent transcription start sites. When the FlrC binding site(s) in the flaA promoter was moved a large distance (285 bp) upstream of the transcription start site of either flaAp or flgKp, high levels of FlrC-dependent transcription resulted, indicating that this binding region functions as an enhancer element. In contrast, the relatively weak FlrC binding site(s) in the flgK promoter failed to function as an enhancer element at either promoter, suggesting that FlrC binding strength contributes to enhancer activity. Our results suggest that the differences in FlrC binding to various flagellar promoters results in the differences in transcription levels that mirror the relative requirement for the flagellar components within the flagellum.

scholarly journals The DNA static curvature has a role in the regulation of the ompS1 porin gene in Salmonella enterica serovar Typhi

Microbiology ◽  
2009 ◽  
Vol 155 (7) ◽  
pp. 2127-2136 ◽  
Author(s):  
Miguel Ángel De la Cruz ◽  
Enrique Merino ◽  
Ricardo Oropeza ◽  
Juan Téllez ◽  
Edmundo Calva
Keyword(s):  
Salmonella Enterica ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Nucleation Site ◽  
Site Directed Mutagenesis ◽  
Dna Curvature ◽  
Structural Topology ◽  
Transcription Process ◽  
Bacterial Genes

The DNA static curvature has been described to play a key role as a regulatory element in the transcription process of several bacterial genes. Here, the role of DNA curvature in the expression of the ompS1 porin gene in Salmonella enterica serovar Typhi is described. The web server mutacurve was used to predict mutations that diminished or restored the extent of DNA curvature in the 5′ regulatory region of ompS1. Using these predictions, curvature was diminished by site-directed mutagenesis of only two residues, and curvature was restored by further mutagenesis of the same two residues. Lowering the extent of DNA curvature resulted in an increase in ompS1 expression and in the diminution of the affinity of the silencer proteins H-NS and StpA for the ompS1 5′ regulatory region. These mutations were in a region shown not to contain the H-NS nucleation site, consistent with the notion that the effect on expression was due to changes in DNA structural topology.

scholarly journals Characterization of CorR, a Transcriptional Activator Which Is Required for Biosynthesis of the Phytotoxin Coronatine

1998 ◽  
Vol 180 (23) ◽  
pp. 6252-6259 ◽  
Author(s):  
Alejandro Peñaloza-Vázquez ◽  
Carol L. Bender
Keyword(s):  
Gene Expression ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Dnase I ◽  
Upstream Region ◽  
Start Site ◽  
Large Area ◽  
Coronafacic Acid

ABSTRACT Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic Pseudomonas syringae. The COR biosynthetic gene cluster in P. syringae pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes: corP,corS, and corR. corS is thought to function as a histidine protein kinase, whereas corP andcorR show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of cfl, a gene located at the 5′ end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a corRmutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (uidA) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5′ end of the cfl upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the cfl transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from −704 to −650 with respect to the cfl transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region forcmaA, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.

scholarly journals Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

1998 ◽  
Vol 180 (9) ◽  
pp. 2367-2372 ◽  
Author(s):  
Sudha A. Chugani ◽  
Matthew R. Parsek ◽  
A. M. Chakrabarty
Keyword(s):  
Binding Site ◽  
Structural Gene ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Dnase I Footprinting ◽  
Gel Shift

ABSTRACT The catBCA operon of Pseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

scholarly journals Positive and Negative Transcriptional Regulation of the Escherichia coli Gluconate Regulon Gene gntTby GntR and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex

1998 ◽  
Vol 180 (7) ◽  
pp. 1777-1785 ◽  
Author(s):  
Norbert Peekhaus ◽  
T. Conway
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Catabolite Repression ◽  
Transcriptional Start Site ◽  
Negative Regulator ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Start Site ◽  
Camp Receptor Protein ◽  
Camp Receptor

ABSTRACT The gntT gene of Escherichia coli is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor ofgntT expression since gluconate is a catabolite-repressing sugar. In a gntR deletion mutant, the expression of a chromosomal gntT::lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT. Indeed, GntR binds to two consensus gnt operator sites; one overlaps the −10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntTexpression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of gntRmutants to the inducer is termed ultrarepression. Transcription ofgntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at −71 upstream of the gntT transcription start site.

scholarly journals The Synthesis of Protein Polymer Conjugates using the Human Regulatory Protein Galectin-3

2020 ◽  
Vol 21 (2) ◽  
Author(s):  
Ling Lin ◽  
Amanda M. Pritzlaff ◽  
Haillie-Ann C. Lower ◽  
Daniel A. Savin
Keyword(s):  
Ligand Binding ◽  
Regulatory Protein ◽  
Site Directed Mutagenesis ◽  
Galectin 3 ◽  
Self Association ◽  
Lectin Protein ◽  
And Control ◽  
Cancerous Cells

Galectin-3 (gal3) is a human lectin protein that is known to interact with extracellular matrix proteins by regulating functions in both healthy and cancerous cells. The goal of this project is to conjugate polymers to gal3 to better study and control its functions in vitro. We hypothesize that a covalently attached polymer will sterically modulate gal3 function. In the project, we created two protein variants with polymer-reactive handles. The first construct is similar to wild-type gal3 with a cysteine in place of the 6th serine (S6C) which was created by site-directed mutagenesis (SDM). Maleimide-terminated polyethylene oxide (PEO, 5000 g/mol) was then attached to this mutant via thiol-Michael addition at the cysteine site. Attachment of polymer to the unstructured N-terminal domain (NTD) may increase the binding of the protein by sterically pulling the NTD away from the carbohydrate recognition domain (CRD). In addition, the NTD, which is implicated in undesired self-association, was removed for the second construct. The gal3 CRD only construct is shown to have a higher solubility in solution and an increased ligand-binding affinity. Ultimately, the two unique constructs will help us understand the structural role of the NTD in gal3 ligand-binding and self-association.

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