A novel type of DNA curvature present in a Clostridium perfringens ferredoxin gene: characterization and role in gene expression

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3083-3091 ◽  
Author(s):  
Masato Kaji ◽  
Osamu Matsushita ◽  
Eiji Tamai ◽  
Shigeru Miyata ◽  
Yuki Taniguchi ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Clostridium Pasteurianum ◽  
Dna Curvature ◽  
Combined Effects ◽  
Gene Characterization

This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the −35 region. The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C. perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively. DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions −69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C. perfringens transformant. Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc. Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter. Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes. These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.

scholarly journals Thymine at —5 Is Crucial for cpc Promoter Activity of Synechocystis sp. Strain PCC 6714

2003 ◽  
Vol 185 (21) ◽  
pp. 6477-6480 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Shoko Fujiwara ◽  
Ryohei Tanigawa ◽  
Kan Tanaka ◽  
Takatsugu Hirokawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Pcc 7942 ◽  
Rna Polymerase Promoter ◽  
Synechocystis Sp

ABSTRACT The levels of transcripts of the cpc operon were highly reduced in a PD-1 mutant of cyanobacterium Synechocystis sp. strain PCC 6714. This was due to a substitution of C for T that occurred at 5 bp upstream of the transcription initiation site of the cpc operon. Any substitution for T at the −5 position drastically reduced both in vivo and in vitro promoter activity in cyanobacterium Synechococcus sp. strain PCC 7942 but not the in vivo activity in Escherichia coli. This suggests that the requirement of −5T appears to be specific for a cyanobacterial RNA polymerase-promoter combination.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

scholarly journals α1-Adrenergic stimulation of FGF-2 promoter in cardiac myocytes and in adult transgenic mouse hearts

1999 ◽  
Vol 276 (3) ◽  
pp. H826-H833 ◽  
Author(s):  
Karen A. Detillieux ◽  
Johanna T. A. Meij ◽  
Elissavet Kardami ◽  
Peter A. Cattini
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Intraperitoneal Administration ◽  
Initiation Site ◽  
Neonatal Rat ◽  
Transcriptional Level ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Neonatal Rat Cardiomyocytes ◽  
Stimulation Of

Fibroblast growth factor (FGF-2), a mitogenic, angiogenic, and cardioprotective agent, is reported to be released from the postnatal heart by a mechanism of transient remodeling of the sarcolemma during contraction. This release can be increased with adrenergic stimulation. RNA blotting was used to assess whether FGF-2 synthesis in neonatal rat cardiomyocytes might also be regulated by adrenergic stimulation. FGF-2 RNA levels were increased after treatment with norepinephrine for 6 h or with the α-adrenergic agonist phenylephrine for 48 h. To assess an effect on transcription, neonatal rat cardiomyocytes were transfected with a hybrid rat FGF-2 promoter/luciferase gene (−1058FGFp. luc) and treated with norepinephrine or phenylephrine for 6 or 48 h, respectively. FGF-2 promoter activity was increased two- to sevenfold in an α1-specific manner. Putative phenylephrine-responsive elements (PEREs) were identified at positions −780 and −761 relative to a major transcription initiation site. However, deletion analysis of −1058FGFp. luc showed that the phenylephrine response was independent of the putative PEREs, cell contraction, and Ca2+ influx. In transgenic mice expressing −1058FGFp. luc, a significant three- to sevenfold stimulation of FGF-2 promoter activity was detected in the hearts of two independent lines 6 h after intraperitoneal administration of phenylephrine (50 mg/kg). This increase was still apparent at 24 h but was not detected at 48 h posttreatment. Analysis of FGF-2 mRNA in normal mouse hearts revealed accumulation of the 6.1-kb transcript at 24 h. Control of local FGF-2 synthesis at the transcriptional level through adrenergic stimulation may be important in the response to injury as well as in the maintenance of a healthy myocardium.

scholarly journals Transcription of Drosophila Troponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

2004 ◽  
Vol 15 (3) ◽  
pp. 1185-1196 ◽  
Author(s):  
María-Cruz Marín ◽  
José-Rodrigo Rodríguez ◽  
Alberto Ferrús
Keyword(s):  
Transcription Initiation ◽  
Troponin I ◽  
Regulatory Element ◽  
Expression Patterns ◽  
In Silico Analysis ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Upstream Regulatory Element ◽  
Transgenic Lines

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

scholarly journals Structure of African Swine Fever Virus Late Promoters: Requirement of a TATA Sequence at the Initiation Region

2000 ◽  
Vol 74 (17) ◽  
pp. 8176-8182 ◽  
Author(s):  
Ramón García-Escudero ◽  
Eladio Viñuela
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
African Swine Fever Virus ◽  
Point Mutations ◽  
African Swine Fever ◽  
Initiation Site ◽  
Fever Virus ◽  
Transcriptional Initiation ◽  
Viral Promoters ◽  
Tata Sequence

ABSTRACT A number of mutations, including deletions, linker scan substitutions, and point mutations, were performed in the promoter of the late African swine fever virus (ASFV) gene coding for the capsid protein p72. The consequences of the mutations in terms of promoter activity were analyzed by luciferase assays using plasmids transfected into infected cells. The results showed that the promoter function is contained between nucleotides −36 and +5 relative to the transcription initiation site. Moreover, two major essential regions for promoter activity, centered at positions −13 and +3, were located along the 41-bp sequence, the latter mapping in the transcription start site. Sequence alignment with other ASFV late promoters showed homology in the region of transcriptional initiation, where the presence of the sequence TATA was observed in most of the promoters. Substitution of these four residues in three other late viral promoters strongly reduced their respective activities. These results show thatcis-acting control elements of ASFV p72 gene transcription are restricted to a short sequence of about 40 bp and suggest that transcription of late genes is initiated around a TATA sequence that would function as an initiator element.

Characterization of a chicken retinoid X receptor-γ gene promoter and identification of sequences that direct expression in retinal cells

2000 ◽  
Vol 347 (2) ◽  
pp. 485-490
Author(s):  
Clara AMEIXA ◽  
Paul M. BRICKELL
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Neural Retina ◽  
Specific Protein ◽  
Retinoid X Receptor ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Enhancer Element ◽  
Extracellular Signals

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-γ (RXR-γ2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-γ genomic clone containing the RXR-γ2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-γ2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-γ2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

scholarly journals Interactions of the Antizyme AtoC with Regulatory Elements of the Escherichia coli atoDAEB Operon

2007 ◽  
Vol 189 (17) ◽  
pp. 6324-6332 ◽  
Author(s):  
Meropi K. Matta ◽  
Efthimia E. Lioliou ◽  
Cynthia H. Panagiotidis ◽  
Dimitrios A. Kyriakidis ◽  
Christos A. Panagiotidis
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Integration Host Factor ◽  
Chemical Mutagenesis ◽  
Dual Function

ABSTRACT AtoC has a dual function as both an antizyme, the posttranslational inhibitor of polyamine biosynthetic enzymes, and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (the atoDAEB operon). We have previously shown that AtoC is the response regulator of the AtoS-AtoC two-component signal transduction system that activates atoDAEB when Escherichia coli is exposed to acetoacetate. Here, we show that the same cis elements control both promoter inducibility and AtoC binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC to the predicted DNA region in vivo. DNase I protection footprinting analysis revealed that AtoC binds two 20-bp stretches, constituting an inverted palindrome, that are located at −146 to −107 relative to the transcription initiation site. Analyses of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC binding for the inducibility of the promoter by acetoacetate and the σ54 dependence of atoDAEB expression. The integration host factor was also identified as a critical component of the AtoC-mediated induction of atoDAEB.

scholarly journals Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6191-6200 ◽  
Author(s):  
Yukako Yamabe ◽  
Akira Shimamoto ◽  
Makoto Goto ◽  
Jun Yokota ◽  
Minoru Sugawara ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Control Element ◽  
Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

scholarly journals Identification of a Previously Unrecognized Promoter That Drives Expression of the UXP Transcription Unit in the Human Adenovirus Type 5 Genome

2010 ◽  
Vol 84 (21) ◽  
pp. 11470-11478 ◽  
Author(s):  
Baoling Ying ◽  
Ann E. Tollefson ◽  
William S. M. Wold
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Mrna Level ◽  
A549 Cells ◽  
Initiation Site ◽  
Transcription Unit ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Ccaat Box

ABSTRACT We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp −158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.

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