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 herpes simplex virus latency-associated transcript (LAT) gene is the only viral gene that shows substantial transcriptional activity during neuronal latency. The LAT RNA produced is antisense to the mRNA of the immediate early gene ICP0, partially overlaps the ICP0 mRNA, and is suspected of playing some role in latency. Sequence analysis of the region 5' to the reported transcription start site has not revealed any high consensus RNA polymerase II promoter elements. Nonetheless, LAT RNA is transcribed in low abundance during acute infection in tissue culture. As the initial step in mapping the promoter for this latency-associated gene, we analysed the ability of different regions of the LAT gene to drive the transcription of an indicator gene in vitro. Using chloramphenicol acetyltransferase (CAT) assays, we found that the genomic region between -940 and -662 nucleotides upstream of the transcription start site of the LAT gene was most efficient at directing transcription of the indicator CAT gene in Vero cells. This suggests that the LAT promoter, or at least the promoter controlling transcription of this gene during acute infection in tissue culture, may have an unusual location of more than 662 nucleotides upstream from the reported start of RNA transcription.
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PMID:In vitro promoter activity associated with the latency-associated transcript gene of herpes simplex virus type 1. 254 85

In order to understand the process of transcription termination by eukaryotic RNA polymerase II, the transcription termination region of the advenovirus 2 major late transcription unit was analysed in a transient transfection system. Previously, it had been demonstrated that the entire sequence from map units (m.u.) 97.1 to 100 of the adenovirus 2 genome terminates transcription when inserted into the 5' or 3' untranslated sequences of the chloramphenicol acetyltransferase gene. Using subclones and Bal 31 deletion mutants of the termination region, we have shown that the termination region consists of multiple elements each capable of inhibiting gene expression independently. A DNA sequence analysis reveals the presence of a highly repetitive A-rich sequence motif throughout the entire termination region. The data suggest that the A-rich motif may mediate the transcription termination process.
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PMID:The transcription termination region of the adenovirus 2 major late transcript contains multiple functional elements. 265 32

Eight eukaryotic promoters have been tested for their activity in vivo in Escherichia coli. The rat beta-actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) and hepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by primer extension RNA analysis. All eight promoter-chloramphenicol acetyltransferase constructs produce chloramphenicol acetyltransferase activity with the following relative strengths: RSV LTR greater than rat beta-actin greater than rat insulin I greater than rat amylase greater than hepatitis B virus precore greater than human insulin greater than rat chymotrypsin B greater than mouse metallothionein I. A primer extension analysis indicates that transcription from the RSV LTR, rat insulin I, and rat beta-actin promoters initiates at the sites expected for eukaryotic rather than prokaryotic promoters. Thus the site of initiation is determined by the DNA sequence rather than by the RNA polymerase.
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PMID:Eukaryotic promoters drive gene expression in Escherichia coli. 268 Nov 82

The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems. Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli. A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator (lacO) was cloned at various distances downstream from the transcription start point (tsp) of the T3 and T7 promoters. The ability of lac repressor to prevent transcription from the phage promoters in vitro was dependent on the position of the operator. Efficient repression was observed when the center of the operator was placed between +14 and +27 (+1 being the tsp), whereas the repressor had little effect when bound to operators centered at +64. For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase. As with in vitro studies, high levels of repression (greater than 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-beta-D-thiogalactopyranoside. When the T3 promoter-lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction. In such a system, we observed a greater than 10(5)-fold increase in CAT activity within 30 min after induction. This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells.
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PMID:Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system. 269 10

Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.
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PMID:Studies on the mechanism of translational enhancement by the 5'-leader sequence of tobacco mosaic virus RNA. 284 Nov 27

The gene for rat cholecystokinin (CCK) was isolated from a rat genomic DNA library. The transcription unit spans 7 kilobases and is interrupted by two introns. The initiator methionine codon lies 2 bases into exon 2; therefore, exon 1 is a noncoding exon. The transcription initiation site was determined using avian myeloblastosis reverse transcriptase, a cDNA primer, and mRNA isolated from a rat medullary thyroid carcinoma. A "TATA"-like sequence precedes the transcription initiation site at position -34. The polyadenylation site for the gene was mapped by a nuclease protection assay using a cRNA generated by transcription of the exon 3 region of the CCK gene with SP6 bacteriophage RNA polymerase. The sequence AT-TAAA is found 22 bases 5' to the site determined to be the polyadenylation addition site. Two regions of simple repetitive DNA occur within the CCK lambda clone, one within intron 2 and the other 4 kilobases 3' to the gene. Sequence analysis of the repetitive element 3' distal to the gene revealed two copies of the sequence 5'-(AC)n-3', where n is 22 and 25. A 114-base pair sequence of predominantly repeating purine-pyrimidine nucleotides separates these two d(AC) repeats. Transcriptional control elements were investigated by fusing regions of the CCK gene to the structural gene encoding chloramphenicol acetyltransferase. Promoter activity was determined by transfecting COS-7 cells with plasmids containing the gene fusions, followed by determining chloramphenicol acetyltransferase activity in cellular extracts. The region necessary for expression of the CCK gene fusions in COS-7 cells is within 144 bases 5' to the initiation of transcription.
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PMID:A gene encoding rat cholecystokinin. Isolation, nucleotide sequence, and promoter activity. 298 40

We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta-globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.
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PMID:Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences. 301 46

A series of mutations, including 5' and 3' deletions, as well as insertions were introduced into the 5' flanking nucleotide sequence of a vaccinia virus late gene. This DNA has been shown previously to contain all the necessary elements for correct regulation of the gene most probably transcribed by the viral RNA polymerase. To facilitate the assays, the mutated DNA was fused to the chloramphenicol acetyltransferase gene and inserted into the genome of live vaccinia virus. The effects of the mutations on expression of the chimeric gene were studied by both enzyme assays and nuclease S1 analysis. The results showed that 5' deletions up to about 15 bp from the putative initiation site of transcription still yielded high levels of gene expression. All mutations, however, that deleted the authentic late mRNA start site, abolished promoter activity.
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PMID:Functional analysis of the 5' flanking sequence of a vaccinia virus late gene. 301 74

Human T-cell leukemia virus type I (HTLV-I) contains the pX sequence which codes for the trans-activator of the long terminal repeat (LTR) and is thus postulated to be associated with leukemogenesis in adult T-cell leukemia. Overlapping open reading frames (ORF) in the pX sequence were recently found to code for p27x-III and p21x-III by ORF III, in addition to p40x coded for by ORF IV. The mechanism of expression of these newly identified proteins and their possible association with trans-activation were studied. On transfection of an expression plasmid that contains a cDNA sequence of the pX mRNA, products from both ORFs III and IV were detected in the cells. The RNA was synthesized in vitro from the cDNA clone by SP6 RNA polymerase and translated in a rabbit reticulocyte lysate. As translation products, two proteins, p27x-III and p21x-III, were detected in addition to p40x. Elimination of the first and second ATG codons in ORF III resulted in loss of the ability to code for p27x-III and p21x-III, respectively, which indicated that the translations from these two ATG codons were independent. A mutant that lacked both ATG codons was fully active in trans-activation of chloramphenicol acetyltransferase gene expression directed by the LTR. These results indicate that a 2.1-kilobase pX mRNA of HTLV-I independently encodes three proteins, p40x, p27x-III, and p21x-III, by different ORFs and that the last two proteins are not involved in trans-activation of the unintegrated LTR.
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PMID:A single species of pX mRNA of human T-cell leukemia virus type I encodes trans-activator p40x and two other phosphoproteins. 302 74

We have analyzed the effect of insertion mutants between the simian virus 40 (SV40) 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4, 42, or 90 bp of DNA at the SV40 NcoI site (map position 37) has been analyzed for its effect on expression of the SV40 early gene and positioning of the RNA 5' ends. Insertion of 4 bp reduced SV40 early promoter-dependent chloramphenicol acetyltransferase (CAT) expression by six- to eightfold. Increasing the size of the insertion to 42 or 90 bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late-early (LE) RNA synthesis was observed. Insertion of 42 or 90 bp of DNA resulted in a decrease of EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA-directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats or the 72-bp repeats or both. That LE RNA levels in pJI1-in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control and probably prefers a 5' initiation codon proximal to that of the CAT gene.
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PMID:Spacing between simian virus 40 early transcriptional control sequences is important for regulation of early RNA synthesis and gene expression. 302 82


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