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)

The nucleotide sequence of the bgaB gene, which encodes the thermostable beta-galactosidase I of Bacillus stearothermophilus, and its flanking region was determined. A 2,016-base-pair open reading frame observed was concluded to be for beta-galactosidase I (Mr 78,051) from observations that the amino acid composition of the enzyme and the sequence of 14 amino acids from the amino-terminus of the enzyme coincided with those deduced from this open frame. A 107-base-pair HaeIII-AluI fragment just upstream of the estimated Shine-Dalgarno sequence of the bgaB gene had promoter activity toward cat-86 (chloramphenicol acetyltransferase gene) and produced the enzyme at a level equivalent to 7% of the total cellular protein of B. subtilis. From the base sequence of this DNA region and the transcriptional start site determined by S1 nuclease mapping, the -35 and -10 sequences are estimated to be TTGACA and TAATTT, respectively, which are similar to the consensus sequence of B. subtilis sigma 43 RNA polymerase.
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PMID:Structure of a beta-galactosidase gene of Bacillus stearothermophilus. 308 88

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.
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PMID:Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. 309 28

The Escherichia coli lacUV5 promoter is used inefficiently by the major vegetative form of Bacillus subtilis RNA polymerase, despite very close adherence in the -35 and -10 regions to consensus sequences for promoters recognized by this enzyme. To select derivatives of this promoter with increased activity in B. subtilis, the lacUV5 promoter was fused to a promoter-less chloramphenicol acetyltransferase gene and mutagenized by passage through an E. coli mutD5 mutator strain. Derivatives that conferred resistance to chloramphenicol in B. subtilis were isolated. Twenty-three independent isolates each contained single mutations in the 207 bp lac fragment. These mutations, which were in ten different positions, fell in two clusters. One set of mutations, located between positions -18 and -14, resulted in greater homology to a consensus sequence previously noted for this region in B. subtilis vegetative promoters. The remaining mutations were located near the transcription initiation site. The effects of these mutations and additional mutations constructed by oligonucleotide-directed mutagenesis on expression in B. subtilis and E. coli was determined by measurements of chloramphenicol acetyltransferase activities directed by these promoters. While most mutations had little effect on expression in E. coli, the increase in activity in B. subtilis was as much as 28-fold.
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PMID:Mutations of the Escherichia coli lacUV5 promoter resulting in increased expression in Bacillus subtilis. 312 85

The effect of DNA upstream of the -35 region on promoter function was examined using two promoters isolated from the Bacillus subtilis bacteriophage SP82. The affinity of RNA polymerase for the two promoters in vitro differed significantly. For each promoter the nucleotide sequence of the upstream DNA was characterized by the presence of successive runs of adenines with a 10-11-base pair periodicity. DNA fragments with the polyadenine-containing upstream DNA displayed aberrant electrophoretic mobilities when analyzed on polyacrylamide gels indicative of curved DNA. A series of mutant promoters in which the upstream DNA was deleted or altered was constructed. The curved DNA upstream of the -35 region was required for efficient RNA polymerase binding. Decreased in vitro transcription observed when the upstream DNA was deleted could be partially restored if the template was negatively supercoiled. Measurements of chloramphenicol acetyltransferase specific activity from B. subtilis strains carrying transcriptional fusions indicate that the curved upstream DNA stimulated transcription from the promoter with the weaker affinity for RNA polymerase. The curved DNA reduced the in vivo activity of the promoter with the strong affinity for RNA polymerase. One function of the curved upstream DNA may be to provide RNA polymerase-promoter interactions that facilitate open complex formation.
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PMID:Effect of polyadenine-containing curved DNA on promoter utilization in Bacillus subtilis. 313 65

DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.
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PMID:Human beta-polymerase gene. Structure of the 5'-flanking region and active promoter. 318 28

We characterized the transcription termination region of the chicken beta H-globin gene. First we located the region by nuclear runon transcription in vitro. Then we sequenced and subcloned it into a chloramphenicol acetyltransferase (CAT) expression vector for assay in vivo. The region of beta H termination contains two interesting elements located about 1 kilobase downstream of the beta H gene poly(A) site. Either element alone can block CAT expression if inserted between the promoter and the poly(A) site of the cat gene in pRSVcat. The first element in the termination region is an unusually large inverted repeat in the DNA (delta G = -71 kcal). The second element, 200 base pairs further downstream, is an RNA polymerase II promoter which directs transcription back upstream on the complementary strand. This transcription converges on and collides with that from the beta H gene at or near the inverted repeat where transcription from both directions stops. We propose that the inverted repeat is a strong pause site which positions the converging polymerases for mutual site-specific termination.
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PMID:Transcription termination at the chicken beta H-globin gene. 324 59

To examine the RNA polymerase (EC 2.7.7.6) specificity of RNA maturation/utilization and transcriptional enhancement, we constructed a chimeric plasmid (pPolI-CAT) in which a promoter for mouse rRNA gene transcription was placed adjacent the coding sequences for chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). A number of other constructs, including plasmids also containing a murine sarcoma virus enhancer or lacking any natural eukaryotic promoter sequences, were also prepared. In apparent agreement with earlier conclusions that an RNA polymerase I transcript can act as a messenger RNA, transient transfection of mouse L cells with pPolI-CAT yielded both high levels of transcription from the RNA polymerase I promoter and enzymatically active CAT protein. However, further examination revealed that CAT protein is not translated from RNA that begins at the normal rRNA transcription initiation site. Polysomal RNA is devoid of such RNA and instead consists of CAT-encoding transcripts that begin elsewhere in the mouse ribosomal DNA (rDNA) region. Since transcription of these aberrant RNAs is stimulated by the addition of a murine sarcoma virus enhancer segment, they are probably transcribed by RNA polymerase II. Transcripts that map to the authentic rRNA start site are not similarly enhanced. Moreover, unlike the RNAs deriving from the rRNA initiation site, these aberrant RNAs are more stable and the level of translatable CAT transcripts is suppressed by inclusion of larger segments of the rDNA promoter regions. Fortuitously initiated mRNAs are also formed in the absence of any natural eukaryotic promoter sequence. From these data we conclude that there is no evidence that normal RNA polymerase I transcription yields functional mRNA and that transcriptional enhancement appears to be RNA polymerase specific.
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PMID:RNA polymerase specificity of mRNA production and enhancer action. 346 18

The high degree of methylation of the frog virus 3 (FV3) genome suggests that FV3-infected cells are capable of transcribing highly methylated DNA. We tested this hypothesis by assaying the transcriptional activity of adenovirus promoters known to be inhibited by methylation. Plasmid constructs containing the E1a and E2aE promoters of adenovirus type 12 linked to the gene for chloramphenicol acetyltransferase [(CAT) EC 2.3.1.28], when methylated and introduced into eukaryotic cells, promoted CAT synthesis only when the cells were subsequently infected with FV3. Mapping of transcriptional initiation sites revealed that the same sites in the E1a promoter were used for the initiation of transcription in uninfected and infected cells. Moreover, Southern blots showed that transfected plasmid DNA from FV3-infected cells was not demethylated. The absence of CAT-specific RNA in transfected cells infected with FV3 in the presence of protein synthesis inhibitors demonstrated that a virus-induced protein was responsible for the trans-activation. Inhibition of transcription from the methylated template by alpha-amanitin indicated that a functional host RNA polymerase II is required for transcription of methylated DNA in FV3-infected cells. The virus-induced trans-acting protein presumably alters either host RNA polymerase II or the methylated DNA template to allow transcription from the methylated adenovirus promoters.
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PMID:Trans-activation of a methylated adenovirus promoter by a frog virus 3 protein. 346 92

A plasmid containing 78 bp of the promoter region of the immediate-early frog virus 3 (FV3) gene ICR 169 placed 5' to the coding sequences for chloramphenicol acetyltransferase (CAT) can only be induced to synthesize CAT after transfection in the presence of FV3. To determine what DNA sequences in the promoter were required for virus-induced transcription, I used site-directed mutagenesis to construct deletions and point mutations throughout the promoter region. The mutant promoters were then analyzed for their ability to be induced by FV3. Deletion of 27 bp from the 5' end of the promoter had little effect on FV3-induced CAT synthesis. Although deletion of, and point mutations within, the 7-bp TATA-like box reduced CAT synthesis to 16-50% of that obtained with the wild-type promoter, only deletion of the 7-bp sequence caused a detectable shift of the transcription start site, indicating that the function of the AT-rich region is to position the RNA polymerase. The most significant reduction in CAT synthesis--to 1.5% of wild-type--occurred after deletion of the 23-bp immediately 5' to the TATTTTA box, which marks this 23-bp sequence as the critical cis-regulatory element for FV3 trans-activation.
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PMID:DNA sequences required for trans-activation of an immediate-early frog virus 3 gene. 347 93

The nucleotide sequence of the inducible chloramphenicol acetyltransferase gene (cat) of Staphylococcus aureus plasmid pC221 has been determined. The deduced primary structure for the 215 residue polypeptide (25.9 kDa) is in agreement with partial amino acid sequence data on the purified protein, previously designated as the type C variant of CAT. In common with the inducible cat elements of pC194 and B. pumilus, the 5' non-coding region of the cat of pC221 contains an inverted complementary repeat ('stem-loop' or 'hairpin') which may sequester the predicted ribosome bonding site of the mRNA. The likely transcription initiation site has been determined in vitro using purified B. subtilis RNA polymerase. Recombinant plasmids carrying the cat of pC221 on a 1156 bp TaqI fragment are expressed inefficiently in Escherichia coli, wherein induction is both poor and orientation-specific.
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PMID:Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221. Nucleotide sequence analysis and expression studies. 385 95


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