Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A plasmid carrying a CRP-dependent promoter fused to the lac structural genes was manipulated to construct a set of spacing mutants that have varying lengths between the CRP binding site and the -35 region. The lengths of the spacer were changed over 45 bp by inserting or deleting nucleotides. DNase I footprinting analysis revealed that the spacer length did not affect the binding of cAMP-CRP to the CRP site. The effect of the spacer length on transcription activation by cAMP-CRP was tested in vivo by beta-galactosidase and quantitative S1 assays with crp+ and delta crp cells harboring plasmids. Insertions or deletions of non-integral helical turns, which displace the CRP site onto the opposite face of DNA helix compared to the original promoter, eliminated completely the activation of transcription. In contrast, changing the spacer length by integral helical turns allowed the promoter to respond to CRP, although the degree of activation varied with the length of the spacer. We conclude that stereospecific positioning of CRP and RNA polymerase on the DNA helix is strictly required for CRP action. The data support a model that CRP stimulates transcription by directly contacting RNA polymerase.
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PMID:Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity. 217 26

The OmpR binding sequence (OBS) in the upstream region of the ompF promoter of Escherichia coli was fused to 27 synthetic promoters. Transcription from a number of weak promoters, regardless of their sequences, was dramatically activated in the presence of OmpR, a transcriptional activator. In vivo DNA footprinting revealed that OmpR enhanced the binding of RNA polymerase to the promoters. This enhancement was essential for transcription of weak promoters, while OmpR binding to the OBS fused to a strong promoter was inhibitory for transcription. These results indicate that OmpR stabilizes the formation of an RNA polymerase-promoter complex, possibly a closed promoter complex, and that a transcription activator can serve not only as a positive but also as a negative regulator for gene expression.
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PMID:Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription. 219 74

We show that the repetitive 140-base-pair (bp) elements present in the spacer of mouse rRNA genes function as enhancers for RNA polymerase I. Attachment of these elements to the rDNA promoter stimulates rRNA synthesis both in vivo and in vitro. The cis-activating effect of the spacer repeats is orientation-independent and increases with increasing numbers of the 140-bp elements. Competition experiments demonstrate that the spacer repeats bind one or more of the transcription factors interaction with the rDNA promoter. Both the 140-bp elements and the core promoter act cooperatively and thus are functionally linked. The 60/81-bp enhancer repeats from Xenopus laevis rDNA compete for a murine transcription factor(s) and stimulate transcription often fused to the mouse rDNA promoter. The results indicate that despite the marked species specificity of rDNA transcription initiation, common factors may interact with both the rDNA promoter and the enhancer.
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PMID:A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system. 221 83

Experimental evidence suggests that centromere arrangement is relevant to the expression of ribosomal genes in murine Sertoli cells. Nuclei endowed with a nucleolus inactive in rRNA synthesis presented several clusters, each containing a bunch of individual centromeres. RNA polymerase I was not cytochemically detected in the nucleolar structure, which contained only small amounts of fibrillarin. In the course of nucleolar activation, the centromeres within the separate clusters became fused into larger centromeric bodies. Synthesis of precursor rRNAs and their processing were visualized by strong nucleolar fluorescence signals using antibodies to RNA polymerase I and fibrillarin.
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PMID:Nucleolar transcriptional activity in mouse Sertoli cells is dependent on centromere arrangement. 222 47

Promoters recognized by RNA polymerase III were used to direct synthesis of RNAs of opposite polarity to the 5' end of the mRNA for the large T-antigen of SV40. A construct was made utilizing the adenovirus (human type II) VA1 gene promoter linked to 163 bp of SV40 DNA sequences cloned in antisense orientation relative to the promoter. The SV40 sequence corresponds to the 5' end of the large T-antigen gene. In addition to the antisense constructs control plasmids were utilized which either lacked both promoter and SV40 elements, lacked RNA polymerase III promoter elements but contained SV40 sequences or contained the VA1 gene promoter fused to SV40 sequences in the sense orientation. The function of the various gene fusions was demonstrated in an in vitro transcription system and in vivo by S1 nuclease 5' end mapping following transfection into COS1 cells. Cotransfection of COS1 cells with the 'antisense' gene and a plasmid containing an SV40 origin of replication resulted in a substantial transient inhibition of SV40-replicon function when compared to control determinations (50% to nearly complete inhibition of large T-antigen dependent DNA replication for 18-36 h). These results show that an antisense RNA generated by RNA polymerase III can effectively block expression of a chromosomally located gene.
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PMID:Inhibition of SV40 replicon function by engineered antisense RNA transcribed by RNA polymerase III. 244 65

Promoters which function in Gram-positive organisms show, with few exceptions, remarkable conservation of sequences identical with those in Escherichia coli. An E. coli system was tested to select putative promoters of two anaerobes, the Gram-positive Clostridium absonum and the Gram-negative Bacteroides thetaiotaomicron. Random restriction fragments of chromosomal DNA from these organisms were fused to the galactokinase (galK) gene of E. coli within a plasmid vector. Approximately 10% of these fragments functioned as promoters in E. coli, and a broad range of activities was evident. A single 88 base pair (bp) C. absonum DNA fragment yielded, in the E. coli plasmid vector, approximately the same high activity as that provided by the E. coli galK promoter. Sequence analysis of this fragment showed typical -35 and -10 sequences, with about five -10-like sequences closely flanking each other, some overlapping, and this appears to result in multiple start sites for transcription. The transcriptions of E. coli plasmid fragments in vitro with both E. coli RNA polymerase and C. absonum RNA polymerase showed pairs of transcripts corresponding to two start sites. By colony hybridization with the 88 bp fragment, radioactively labelled, as a probe, a 4.2 kilobase segment of C. absonum chromosomal DNA containing the 88 bp fragment was isolated. About 375 bp of this fragment was sequenced. A putative Shine-Dalgarno sequence and ATG start site were detected, followed by an opening reading frame. Using a sequence about 100 bp downstream from the 88 bp sequence, a 17-base oligonucleotide was synthesized to serve as a primer. With C. absonum RNA as a template, a reverse transcriptase primer extension assay located a pair of transcription start sites just downstream from the 88 bp sequence, proving that the 88 bp sequence functions as a promoter in C. absonum.
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PMID:Isolation of promoters from two anaerobic bacteria. 245 83

We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spo0H, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spo0H encodes a sigma factor of RNA polymerase, sigma H, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by sigma H (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn917lac, in the presence and absence of sigma H. These genes had lower levels of expression in the absence of sigma H than in the presence of sigma H. Patterns of expression of the csh genes during growth and sporulation in wild-type and spo0H mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn917lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spo0H mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spo0H in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.
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PMID:Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis. 250 32

The fumarase gene (citG) of Bacillus subtilis is transcribed from two promoter regions, citGp1 and citGp2 (P1 and P2); the P2 promoter is used by the E sigma H form of RNA polymerase. In order to study the role of P1 and P2 in citG expression, the promoter region and various deletion derivatives that effectively separate P1 and P2 were fused to the Escherichia coli beta-galactosidase gene (lacZ) and introduced into the chromosome in single copy at the amyE locus. P1 functioned to provide a relatively low and stable basal level of fumarase activity throughout growth. In contrast, P2 activity was found to vary over at least a 50-fold range and was responsible for regulating fumarase activity during growth and sporulation in a rich medium and in response to changes in carbon source. To further investigate the role of sigma H in fumarase regulation, citGp2-lacZ fusions were introduced into a strain in which the expression of the chromosomal spoOH gene was under the control of the isopropylthiogalactopyranoside-inducible spac promoter. Induction of pspac did not lead to P2 induction, suggesting that citG expression is not regulated at the level of spoOH transcription.
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PMID:Role of sigma H in expression of the fumarase gene (citG) in vegetative cells of Bacillus subtilis 168. 250 23

Transcription of the yeast CYC1 promoter fused to a sequence lacking guanosine residues provided a rapid, sensitive assay of initiation by RNA polymerase II in yeast extracts. Initiation was enhanced by yeast and mammalian activator proteins. The adenoviral major late promoter fused to the G-minus sequence was transcribed in yeast extracts with an efficiency comparable to that observed in HeLa extracts, showing that promoters as well as transcription factors are functionally interchangeable across species. Initiation occurred at different sites, approximately 30 and 63 to 69 base pairs downstream of the TATA element of the adenoviral promoter in HeLa and yeast extracts, respectively, distances characteristic of initiation in the two systems in vivo. A component of the transcription system and not the promoter sequence determines the distance to the initiation site.
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PMID:Initiation by yeast RNA polymerase II at the adenoviral major late promoter in vitro. 251 Feb 98

The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue. The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate. The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257. The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity. The kinase domain also behaved as an oligomeric protein during gel filtration. The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.
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PMID:Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli. 255 38


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