scholarly journals Regulation of the furA and catC Operon, Encoding a Ferric Uptake Regulator Homologue and Catalase-Peroxidase, Respectively, in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (13) ◽  
pp. 3767-3774 ◽  
Author(s):  
Ji-Sook Hahn ◽  
So-Young Oh ◽  
Jung-Hye Roe
Keyword(s):  
Streptomyces Coelicolor ◽  
Transcriptional Start Site ◽  
Protein Product ◽  
Negative Regulator ◽  
Start Codon ◽  
Multicopy Plasmid ◽  
Ferric Uptake Regulator ◽  
Start Site ◽  
Reading Frame ◽  
Catalase Peroxidase

ABSTRACT We isolated the catC gene, encoding catalase-peroxidase in Streptomyces coelicolor, using sequence homology with the katG gene from Escherichia coli. Upstream of the catC gene, an open reading frame (furA) encoding a homologue of ferric uptake regulator (Fur) was identified. S1 mapping analysis indicated that the furA gene was cotranscribed with the catC gene. The transcriptional start site of the furA-catC mRNA was mapped to the translation start codon ATG of the furA gene. The putative promoter contains consensus −10 and −35 elements similar to those recognized by ςHrdB, the major sigma factor of S. coelicolor. The transcripts were produced maximally at late-exponential phase and decreased at the stationary phase in liquid culture. The change in the amount of mRNA was consistent with that of CatC protein and enzyme activity. When the furA gene was introduced into S. lividans on a multicopy plasmid, the increased production of catC transcripts and protein product at late growth phase was inhibited, implying a role for FurA as the negative regulator of the furA-catC operon. FurA protein bound to its own promoter region between −59 and −39 nucleotides from the transcription start site. The binding affinity of FurA increased under reducing conditions and in the presence of metals such as Ni2+, Mn2+, Zn2+, or Fe2+. Addition of these metals to the growth medium decreased the production of CatC protein, consistent with the role of FurA as a metal-dependent repressor.

scholarly journals Transcriptional Analysis and Regulation of Expression of the ScrFI Restriction-Modification System of Lactococcus lactis subsp.cremoris UC503

2001 ◽  
Vol 183 (15) ◽  
pp. 4668-4673 ◽  
Author(s):  
Derek Butler ◽  
Gerald F. Fitzgerald
Keyword(s):  
Transcriptional Start Site ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Northern Analysis ◽  
Start Site ◽  
Regulation Of Expression ◽  
Modification System ◽  
Reading Frame ◽  
Restriction Modification System ◽  
Restriction Modification

ABSTRACT ScrFI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5′-CC↓ NGG-3′, cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame (orfX) of unknown function is located immediately upstream of these genes. In this study Northern analysis was performed, and it revealed that orfX, scrFIBM, andscrFIR are cotranscribed as a single polygenic mRNA molecule, while scrFIAM is transcribed independently. 5′ extension analysis indicated that the start site for thescrFIAM promoter was a thymine located 4 bp downstream of the −10 motif. The transcriptional start site for theorfX promoter was also found to be a thymine which is more atypically located 24 bp downstream of the −10 motif proximal to the start codon. A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M.ScrFIA). In order to determine if this motif played a role in regulation of the ScrFI locus, M.ScrFIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the ScrFI operon, one located upstream oforfX and the other located just upstream ofscrFIAM. M.ScrFIA was found to bind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless lacZ gene, and cloned fragments of theScrFI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M.ScrFIA in regulation of both promoters on the ScrFI locus.

scholarly journals Forespore-Specific Expression of Bacillus subtilis yqfS, Which Encodes Type IV Apurinic/Apyrimidinic Endonuclease, a Component of the Base Excision Repair Pathway

2003 ◽  
Vol 185 (1) ◽  
pp. 340-348 ◽  
Author(s):  
Norma Urtiz-Estrada ◽  
José M. Salas-Pacheco ◽  
Ronald E. Yasbin ◽  
Mario Pedraza-Reyes
Keyword(s):  
Bacillus Subtilis ◽  
Excision Repair ◽  
Transcriptional Start Site ◽  
Northern Blotting ◽  
Start Codon ◽  
Start Site ◽  
Specific Expression ◽  
Reading Frame ◽  
Type Iv

ABSTRACT The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the EσG regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the σG regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.

scholarly journals Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

1999 ◽  
Vol 181 (3) ◽  
pp. 849-857 ◽  
Author(s):  
P. Aneja ◽  
T. C. Charles
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Start Codon ◽  
Start Site ◽  
Gene Encoding ◽  
Reading Frame ◽  
Isolation And Characterization

ABSTRACT We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.

scholarly journals Regulation of Expression of the adhE Gene, Encoding Ethanol Oxidoreductase in Escherichia coli: Transcription from a Downstream Promoter and Regulation by Fnr and RpoS

1999 ◽  
Vol 181 (24) ◽  
pp. 7571-7579 ◽  
Author(s):  
Jorge Membrillo-Hernández ◽  
E. C. C. Lin
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Start Site ◽  
Start Codon ◽  
Start Site ◽  
Rnase Iii ◽  
Regulation Of Expression ◽  
Experimental Conditions ◽  
Stem Loop ◽  
Translational Start ◽  
Transcriptional Start Sites

ABSTRACT The adhE gene of Escherichia coli, located at min 27 on the chromosome, encodes the bifunctional NAD-linked oxidoreductase responsible for the conversion of acetyl-coenzyme A to ethanol during fermentative growth. The expression of adhEis dependent on both transcriptional and posttranscriptional controls and is about 10-fold higher during anaerobic than during aerobic growth. Two putative transcriptional start sites have been reported: one at position −292 and the other at −188 from the translational start codon ATG. In this study we show, by using several different transcriptional and translational fusions to the lacZ gene, that both putative transcriptional start sites can be functional and each site can be redox regulated. Although both start sites are NarL repressible in the presence of nitrate, Fnr activates only the −188 start site and Fis is required for the transcription of only the −292 start site. In addition, it was discovered that RpoS activatesadhE transcription at both start sites. Under all experimental conditions tested, however, only the upstream start site is active. Available evidence indicates that under those conditions, the upstream promoter region acts as a silencer of the downstream transcriptional start site. Translation of the mRNA starting at −292, but not the one starting at −188, requires RNase III. The results support the previously postulated ribosomal binding site (RBS) occlusion model, according to which RNase III cleavage is required to release the RBS from a stem-loop structure in the long transcript.

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 Characterization of the promoter of human adipocyte hormone-sensitive lipase

1997 ◽  
Vol 328 (2) ◽  
pp. 453-461 ◽  
Author(s):  
Jacques GROBER ◽  
Henrik LAURELL ◽  
Régis BLAISE ◽  
Béatrice FABRY ◽  
Stéphane SCHAAK ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Rnase H ◽  
Hormone Sensitive Lipase ◽  
Start Codon ◽  
Start Site ◽  
Rnase Protection ◽  
Human Adipocyte ◽  
Hormone Sensitive

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5ʹ untranslated region (5ʹ-UTR). A single 5ʹ-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5ʹ-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5ʹ-UTR is mutually exclusive. The short and long 5ʹ-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5ʹ-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

scholarly journals The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

2002 ◽  
Vol 184 (6) ◽  
pp. 1801-1805 ◽  
Author(s):  
Brigitta Kurenbach ◽  
Dagmar Grothe ◽  
María Eugenia Farías ◽  
Ulrich Szewzyk ◽  
Elisabeth Grohmann
Keyword(s):  
Amino Acids ◽  
Transcriptional Start Site ◽  
Conjugative Plasmid ◽  
Start Codon ◽  
Start Site ◽  
Gene Encoding ◽  
Cleavage Activity ◽  
Reverse Transcription Pcr ◽  
Single Operon

ABSTRACT The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriTpIP501 . The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg2+ or Mn2+ and is highest at temperatures of between 42 and 45°C.

scholarly journals Upstream Open Reading Frame Mediated Translation of WNK8 Is Required for ABA Response in Arabidopsis

2021 ◽  
Vol 22 (19) ◽  
pp. 10683
Author(s):  
Zhiyong Li ◽  
Yajuan Fu ◽  
Jinyu Shen ◽  
Jiansheng Liang
Keyword(s):  
Open Reading Frame ◽  
Negative Regulator ◽  
Upstream Open Reading Frame ◽  
Start Codon ◽  
Aba Signaling ◽  
Glucose Response ◽  
Reading Frame ◽  
Downstream Target ◽  
Wide Range ◽  
Aba Response

With no lysine (K) (WNK) kinases comprise a family of serine/threonine kinases belonging to an evolutionary branch of the eukaryotic kinome. These special kinases contain a unique active site and are found in a wide range of eukaryotes. The model plant Arabidopsis has been reported to have 11 WNK members, of which WNK8 functions as a negative regulator of abscisic acid (ABA) signaling. Here, we found that the expression of WNK8 is post-transcriptionally regulated through an upstream open reading frame (uORF) found in its 5′ untranslated region (5′-UTR). This uORF has been predicted to encode a conserved peptide named CPuORF58 in both monocotyledons and dicotyledons. The analysis of the published ribosome footprinting studies and the study of the frameshift CPuORF58 peptide with altered repression capability suggested that this uORF causes ribosome stalling. Plants transformed with the native WNK8 promoter driving WNK8 expression were comparable with wild-type plants, whereas the plants transformed with a similar construct with mutated CPuORF58 start codon were less sensitive to ABA. In addition, WNK8 and its downstream target RACK1 were found to synergistically coordinate ABA signaling rather than antagonistically modulating glucose response and flowering in plants. Collectively, these results suggest that the WNK8 expression must be tightly regulated to fulfill the demands of ABA response in plants.

scholarly journals The Gene ssl3076 Encodes a Protein Mediating the Salt-Induced Expression of ggpS for the Biosynthesis of the Compatible Solute Glucosylglycerol in Synechocystis sp. Strain PCC 6803

2010 ◽  
Vol 192 (17) ◽  
pp. 4403-4412 ◽  
Author(s):  
Stephan Klähn ◽  
Antje Höhne ◽  
Elke Simon ◽  
Martin Hagemann
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transcriptional Start Site ◽  
Gene Promoter ◽  
Compatible Solute ◽  
Negative Regulator ◽  
Specific Gene ◽  
Reading Frame ◽  
Low Salt ◽  
Pcc 6803

ABSTRACT Acclimation to high salt concentrations involves concerted changes in gene expression. For the majority of salt-regulated genes, the mechanism underlying the induction process is not known. The gene ggpS (sll1566), which encodes the glucosylglycerol-phosphate synthase responsible for the synthesis of the compatible solute glucosylglycerol (GG), is specifically induced by salt in the cyanobacterial model strain Synechocystis sp. strain PCC 6803. To identify mechanisms mediating this salt-specific gene regulation, the ggpS promoter was analyzed in more detail. 5′ rapid amplification of cDNA ends (5′-RACE) experiments revealed that the adjacent open reading frame (ORF), which is annotated as unknown protein Ssl3076, overlaps with the transcriptional start site of the ggpS gene. Reporter gene expression analyses indicated an essential role for the intact ssl3076 gene in the salt-regulated transcription of a gfp reporter gene. Promoter fragments containing a mutated ssl3076 lost the salt regulation; similarly, a frameshift mutation in ssl3076 resulted in a high level of ggpS expression under low-salt conditions, thereby establishing this small ORF, named ggpR, as a negative regulator of ggpS. Interestingly, small ORFs were also found adjacent to ggpS genes in the genomes of other GG-accumulating cyanobacteria. These results suggest that the GgpR protein represses ggpS expression under low-salt conditions, whereas in salt-shocked and salt-acclimated cells a stress-proportional ggpS expression occurs, leading to GG accumulation.

scholarly journals Regulation of hepA ofAnabaena sp. Strain PCC 7120 by Elements 5′ from the Gene and by hepK

1998 ◽  
Vol 180 (16) ◽  
pp. 4233-4242 ◽  
Author(s):  
Jinsong Zhu ◽  
Renqiu Kong ◽  
C. Peter Wolk
Keyword(s):  
Dna Sequences ◽  
System Analysis ◽  
Transcriptional Start Site ◽  
Constitutive Expression ◽  
Regulatory System ◽  
Nitrogen Deprivation ◽  
Start Site ◽  
Mobility Shift ◽  
Translational Initiation ◽  
Reading Frame

ABSTRACT In Anabaena spp., synthesis of the heterocyst envelope polysaccharide, required if the cell is to fix dinitrogen under aerobic conditions, is dependent on the gene hepA. A transcriptional start site of hepA was localized 104 bp 5′ from its translational initiation codon. A 765-bp open reading frame, denoted hepC, was found farther upstream. Inactivation ofhepC led to constitutive expression of hepA and prevented the synthesis of heterocyst envelope polysaccharide. However, the glycolipid layer of the heterocyst envelope was synthesized. AhepK mutation blocked both the synthesis of the heterocyst envelope polysaccharide and induction of hepA. The predicted product of hepK resembles a sensory protein-histidine kinase of a two-component regulatory system. Analysis of the region between hepC and hepA indicated that DNA sequences required for the induction of hepA upon nitrogen deprivation are present between bp −574 and −440 and between bp −340 and −169 relative to the transcriptional start site ofhepA. Gel mobility shift assays provided evidence that one or more proteins bind specifically to the latter sequence. The Fox box sequence downstream from hepA appeared inessential for the induction of hepA.

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