scholarly journals Analysis of cis- and trans-Acting Factors Involved in Regulation of the Streptococcus mutans Fructanase Gene (fruA)

2002 ◽  
Vol 184 (1) ◽  
pp. 126-133 ◽  
Author(s):  
Zezhang T. Wen ◽  
Robert A. Burne
Keyword(s):  
Streptococcus Mutans ◽  
Catabolite Repression ◽  
Transcription Initiation ◽  
Glucose Repression ◽  
Carbon Sources ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Site Directed Mutagenesis ◽  
Phosphotransferase System ◽  
Insertional Inactivation

ABSTRACT There are two primary levels of control of the expression of the fructanase gene (fruA) of Streptococcus mutans: induction by levan, inulin, or sucrose and repression in the presence of glucose and other readily metabolized sugars. The goals of this study were to assess the functionality of putative cis-acting regulatory elements and to begin to identify the trans-acting factors involved in induction and catabolite repression of fruA. The fruA promoter and its derivatives generated by deletions and/or site-directed mutagenesis were fused to a promoterless chloramphenicol acetyltransferase (CAT) gene as a reporter, and strains carrying the transcriptional fusions were then analyzed for CAT activities in response to growth on various carbon sources. A dyadic sequence, ATGACA(TC)TGTCAT, located at −72 to −59 relative to the transcription initiation site was shown to be essential for expression of fruA. Inactivation of the genes that encode fructose-specific enzymes II resulted in elevated expression from the fruA promoter, suggesting negative regulation of fruA expression by the fructose phosphotransferase system. Mutagenesis of a terminator-like structure located in the 165-base 5′ untranslated region of the fruA mRNA or insertional inactivation of antiterminator genes revealed that antitermination was not a mechanism controlling induction or repression of fruA, although the untranslated leader mRNA may play a role in optimal expression of fructanase. Deletion or mutation of a consensus catabolite response element alleviated glucose repression of fruA, but interestingly, inactivation of the ccpA gene had no discernible effect on catabolite repression of fruA. Accumulating data suggest that expression of fruA is regulated by a mechanism that has several unique features that distinguish it from archetypical polysaccharide catabolic operons of other gram-positive bacteria.

A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes

1991 ◽  
Vol 11 (2) ◽  
pp. 641-654
Author(s):  
C Hinkley ◽  
M Perry
Keyword(s):  
Cell Cycle ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Histone Gene ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequences ◽  
Chromosomal Dna ◽  
Transcriptional Regulatory ◽  
Octamer Motif

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

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 Genomic organization and transcriptional analysis of the human l-glutaminase gene

2003 ◽  
Vol 370 (3) ◽  
pp. 771-784 ◽  
Author(s):  
Cristina PÉREZ-GÓMEZ ◽  
José M. MATÉS ◽  
Pedro M. GÓMEZ-FABRE ◽  
Antonio del CASTILLO-OLIVARES ◽  
Francisco J. ALONSO ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Genomic Organization ◽  
Core Promoter ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Genome Project ◽  
Transcriptional Analysis ◽  
Cdna Clones ◽  
Mobility Shift ◽  
Specific Expression

In mammals, glutaminase (GA) is expressed in most tissues, but the regulation of organ-specific expression is largely unknown. Therefore, as an essential step towards studying the regulation of GA expression, the human liver-type GA (hLGA) gene has been characterized. LGA genomic sequences were isolated using the genome walking technique. Analysis and comparison of these sequences with two LGA cDNA clones and the Human Genome Project database, allowed the determination of the genomic organization of the LGA gene. The gene has 18 exons and is approx. 18kb long. All exon/intron junction sequences conform to the GT/AG rule. Progressive deletion analysis of LGA promoter—luciferase constructs indicated that the core promoter is located between nt −141 and +410, with several potential regulatory elements: CAAT, GC, TATA-like, Ras-responsive element binding protein and specificity protein 1 (Sp1) sites. The minimal promoter was mapped within +107 and +410, where only an Sp1 binding site is present. Mutation experiments suggested that two CAAT recognition elements near the transcription-initiation site (-138 and −87), play a crucial role for optimal promoter activity. Electrophoretic mobility-shift assays confirmed the importance of CAAT- and TATA-like boxes to enhance basal transcription, and demonstrated that HNF-1 motif is a significant distal element for transcriptional regulation of the hLGA gene.

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 cis-acting sequences of the rat troponin I slow gene confer tissue- and development-specific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice.

1993 ◽  
Vol 13 (11) ◽  
pp. 7019-7028 ◽  
Author(s):  
S Banerjee-Basu ◽  
A Buonanno
Keyword(s):  
Skeletal Muscle ◽  
Transgenic Mice ◽  
Transcription Initiation ◽  
Troponin I ◽  
Regulatory Elements ◽  
Initiation Site ◽  
C2c12 Cells ◽  
Fiber Types ◽  
Tibialis Muscle ◽  
Flanking Sequences

Transcription of the genes coding for troponin I slow (TnIslow) and other contractile proteins is activated during skeletal muscle differentiation, and their expression is later restricted to specific fiber types during maturation. We have isolated and characterized the rat TnIslow gene in order to begin elucidating its regulation during myogenesis. Transcriptional regulatory regions were delineated by using constructs, containing TnIslow gene sequences driving the expression of the chloramphenicol acetyltransferase (CAT) reporter gene, that were transiently transfected into undifferentiated and differentiated C2C12 cells. TnIslow 5'-flanking sequences directed transcription specifically in differentiated cells. However, transcription rates were approximately 10-fold higher in myotubes transfected with constructs containing the 5'-flanking sequences plus the intragenic region residing upstream of the translation initiation site (introns 1 and 2), indicative of interactions between elements residing upstream and in the introns of the gene. Deletion analysis of the 5' region of the TnIslow gene showed that the 200 bp upstream of the transcription initiation site is sufficient to confer differentiation-specific transcription in C2C12 myocytes. MyoD consensus binding sites were found both in the upstream 200-bp region and in a region residing in the second intron that is highly homologous to the quail TnIfast enhancer. Transactivation experiments using transfected NIH 3T3 fibroblasts with TnI-CAT constructs containing intragenic and/or upstream sequences and with the myogenic factors MyoD, myogenin, and MRF4 showed different potentials of these factors to induce transcription. Transgenic mice harboring the rat TnI-CAT fusion gene expressed the reporter specifically in the skeletal muscle. Furthermore, CAT levels were approximately 50-fold higher in the soleus than in the extensor digitorum longus, gastrocnemius, or tibialis muscle, indicating that the regulatory elements that restrict TnI transcription to slow-twitch myofibers reside in the sequences we have analyzed.

scholarly journals Regulation and Physiologic Significance of the Agmatine Deiminase System of Streptococcus mutans UA159

2006 ◽  
Vol 188 (3) ◽  
pp. 834-841 ◽  
Author(s):  
Ann R. Griswold ◽  
Max Jameson-Lee ◽  
Robert A. Burne
Keyword(s):  
Streptococcus Mutans ◽  
Transcription Initiation ◽  
Acid Tolerance ◽  
Acid Resistance ◽  
Initiation Site ◽  
Low Ph ◽  
Pathogenic Potential ◽  
Genome Database ◽  
Reading Frame ◽  
Agmatine Deiminase

ABSTRACT We previously demonstrated that Streptococcus mutans expresses a functional agmatine deiminase system (AgDS) encoded by the agmatine-inducible aguBDAC operon (A. R. Griswold, Y. Y. Chen, and R. A. Burne, J. Bacteriol. 186:1902-1904, 2004). The AgDS yields ammonia, CO2, and ATP while converting agmatine to putrescine and is proposed to augment the acid resistance properties and pathogenic potential of S. mutans. To initiate a study of agu gene regulation, the aguB transcription initiation site was identified by primer extension and a putative σ70-like promoter was mapped 5′ to aguB. Analysis of the genome database revealed an open reading frame (SMU.261c) encoding a putative transcriptional regulator located 239 bases upstream of aguB. Inactivation of SMU.261c decreased AgD activity by sevenfold and eliminated agmatine induction. AgD was also found to be induced by certain environmental stresses, including low pH and heat, implying that the AgDS may also be a part of a general stress response pathway of this organism. Interestingly, an AgDS-deficient strain was unable to grow in the presence of 20 mM agmatine, suggesting that the AgDS converts a growth-inhibitory substance into products that can enhance acid tolerance and contribute to the competitive fitness of the organism at low pH. The capacity to detoxify and catabolize agmatine is likely to have major ramifications on oral biofilm ecology.

scholarly journals Heavy Involvement of Stringent Transcription Control Depending on the Adenine or Guanine Species of the Transcription Initiation Site in Glucose and Pyruvate Metabolism in Bacillus subtilis

2010 ◽  
Vol 192 (6) ◽  
pp. 1573-1585 ◽  
Author(s):  
Shigeo Tojo ◽  
Kanako Kumamoto ◽  
Kazutake Hirooka ◽  
Yasutaro Fujita
Keyword(s):  
Transcription Initiation ◽  
Pyruvate Dehydrogenase Complex ◽  
Stringent Response ◽  
Initiation Site ◽  
Phosphotransferase System ◽  
Pyruvate Metabolism ◽  
Transcription Control ◽  
Initiation Rate ◽  
Positive Direction ◽  
Stringent Control

ABSTRACT In B acillus subtilis cells, the GTP level decreases and the ATP level increases upon a stringent response. This reciprocal change in the concentrations of the substrates of RNA polymerase affects the rate of transcription initiation of certain stringent genes depending on the purine species at their transcription initiation sites. DNA microarray analysis suggested that not only the rrn and ilv-leu genes encoding rRNAs and the enzymes for synthesis of branched-chain amino acids, respectively, but also many genes, including genes involved in glucose and pyruvate metabolism, might be subject to this kind of stringent transcription control. Actually, the ptsGHI and pdhABCD operons encoding the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system and the pyruvate dehydrogenase complex were found to be negatively regulated, like rrn, whereas the pycA gene encoding pyruvate carboxylase and the alsSD operon for synthesis of acetoin from pyruvate were positively regulated, like ilv-leu. Replacement of the guanine at position 1 and/or position 2 of ptsGHI and at position 1 of pdhABCD (transcription initiation base at position 1) by adenine changed the negative stringent control of these operons in the positive direction. The initiation bases for transcription of pdhABCD and pycA were newly determined. Then the promoter sequences of these stringent operons were aligned, and the results suggested that the presence of a guanine(s) and the presence of an adenine(s) at position 1 and/or position 2 might be indispensable for negative and positive stringent control, respectively. Such stringent transcription control that affects the transcription initiation rate through reciprocal changes in the GTP and ATP levels likely occurs for numerous genes of B. subtilis.

scholarly journals Identification of a second β-glucoside phosphoenolpyruvate : carbohydrate phosphotransferase system in Corynebacterium glutamicum R

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3652-3660 ◽  
Author(s):  
Yuya Tanaka ◽  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Corynebacterium Glutamicum ◽  
Catabolite Repression ◽  
Gene Disruption ◽  
Single Gene ◽  
Carbon Sources ◽  
Phosphotransferase System ◽  
Carbohydrate Transport ◽  
Encoding Genes ◽  
Redundant Role

The phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) catalyses carbohydrate transport by coupling it to phosphorylation. Previously, we reported a Corynebacterium glutamicum R β-glucoside PTS encoded by bglF. Here we report that C. glutamicum R contains an additional β-glucoside PTS gene, bglF2, organized in a cluster with a putative phospho-β-glucosidase gene, bglA2, and a putative antiterminator, bglG2. While single gene disruption strains of either bglF or bglF2 were able to utilize salicin or arbutin as sole carbon sources, a double disruption strain exhibited defects in utilization of both carbon sources. Expression of both bglF and bglF2 was induced in the presence of either salicin or arbutin, although disruption of bglG2 affected only bglF2 expression. Moreover, in the presence of either salicin or arbutin, glucose completely repressed the expression of bglF but only slightly repressed that of bglF2. We conclude that BglF and BglF2 have a redundant role in β-glucoside transport even though the catabolite repression control of their encoding genes is different. We also show that expression of both bglF and bglF2 requires the general PTS.

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