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)

After NIH3T3 cells constitutively expressing T7 RNA polymerase were transfected (+ Ca.phosphate) with a circular DNA containing the firefly luciferase(Luc)-encoding gene (luc) 3' to the encephalomyocarditis (EMC) virus 5'-untranslated sequence and T7 promoter, Luc protein comprising approx. 20% of total cellular protein was obtained. After similar transfection of an analogous construct containing the lacZ gene into the same cell line, at least 50% of the cells produced beta-galactosidase. Fibroblasts lipofected with uncapped RNA transcripts containing EMC sequence expressed the reporter genes as efficiently as capped transcripts. A novel approach was used to generate RNA transcripts containing poly(A) at its very 3' end. RNA from a luc vector with a poly(A) sequence at the very 3' end produced 20-fold more Luc than the RNA from the same vector with an additional 3' nonpoly(A) sequence. These results suggest that this T7 RNA polymerase expression system will be useful for the efficient production of proteins in mammalian cells.
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PMID:High-efficiency protein synthesis from T7 RNA polymerase transcripts in 3T3 fibroblasts. 166 54

The Escherichia coli treA gene encodes an osmotically inducible periplasmic trehalase. A strain carrying a treA-lacZ transcriptional fusion was constructed. The beta-galactosidase activity produced in this strain growing exponentially in a medium of high osmotic pressure was 10-fold higher than that produced in a medium of low osmotic pressure, demonstrating that treA transcription is osmotically inducible. treA transcriptional induction depends neither on the presence of trehalase itself nor on the synthesis of cytoplasmic trehalose which occurs in response to osmotic stress in wild-type E. coli strains. The treA promoter was identified by S1 nuclease protection. Deletion analysis demonstrated that sequences sufficient for the osmotic induction lie downstream from nucleotide -40 with respect to the transcription start. Transcription initiation at treAp required the presence of a functional sigma 70 subunit of RNA polymerase. treA expression was increased in the presence of a mutation in osmZ, which was previously identified as leading to a partially constitutive expression of the osmotically inducible proU operon.
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PMID:Osmotic induction of the periplasmic trehalase in Escherichia coli K12: characterization of the treA gene promoter. 171 Jul 60

L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced. A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase. These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspH1 mutation at 215 degrees. Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium. In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t1 to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73. The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present. In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript. Depending on the growth medium, the levels of asparaginase and aspartase were from 2- to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspH1 mutation codes for a trans-acting transcriptional regulatory factor. In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by t0 of sporulation but then showed a small increase, which was mirrored by changes in the level of beta-galactosidase from an ansB-lacZ fusion. The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore. However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulation-specific RNA polymerase tested yielded a -73 transcript in vitro.
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PMID:Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase. 171 Oct 29

The effects of mutations in the -10, -35, and Fnr box regions of the narGHJI promoter of Escherichia coli were determined by assaying the expression of beta-galactosidase from narG::lacZ fusion plasmids under aerobic and anaerobic conditions. A 1-base change in the -10 hexamer completely abolished expression, whereas a 3-base change to create the consensus TATAAT resulted in significant aerobic as well as anaerobic expression. A mutation in the putative -35 hexamer did not affect anaerobic expression but reduced aerobic expression from the construction with the -10 consensus sequence. A mutation in the Fnr box severely reduced anaerobic expression but did not affect aerobic expression. When the complete 5' region of the nar operon including the NarL box was present, nitrate stimulated both aerobic and anaerobic expression. Stimulation of expression by nitrate occurred in an fnr mutant but not in a narL mutant. We conclude that the rate of transcription of the nar operon is dependent on two distinct modes of transcription. One mode, which occurs at low levels, depends on the -10 and -35 hexamer sequences and is dramatically enhanced by changing the -10 sequence to the consensus TATAAT. The second depends on the -10 and Fnr box sequences but is independent of the -35 sequence. This second mode occurs at a very high level under anaerobic conditions when Fnr is activated and is also enhanced by changing the -10 sequence to the consensus TATAAT. NarL, activated by nitrate, stimulated both modes of transcription, indicating that it does not act through Fnr but that it directly affects the interaction of RNA polymerase with the promoter.
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PMID:Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli. 173 6

Translational lacZ fusions to forespore genes of Bacillus subtilis were not expressed in spoIIAC (sigma F) or spoIIIE mutants when the lacZ fusions were integrated at the loci of the same genes or at the SP beta locus. However, some of these genes, including gerA, gpr, spoIIIG (sigma G), and sspE, were expressed in spoIIIE mutants and spoIIIE spoIIIG double mutants (but not in spoIIAC mutants) when the lacZ fusions were integrated at the amyE locus. When tested, the beta-galactosidase made in these mutants was found only in the forespore, and the 5' ends of the mRNAs produced in these mutants were identical to those in a Spo+ background. Analysis of the in vitro transcription of forespore genes by RNA polymerase containing sigma F (E sigma F) revealed a direct correlation between good in vitro transcription by E sigma F and expression at the amyE locus in spoIIIE mutants. This result suggests that forespore genes are transcribed by E sigma F in spoIIIE and spoIIIE spoIIIG mutants. Comparison of the promoter regions of genes transcribed well and poorly by E sigma F in vivo and in vitro showed that good transcription by E sigma F was correlated with G residues at positions -15 and -16, a purine residue at position -13, and a T residue at position -7 relative to the start site of transcription. The importance of these residues in sigma F recognition was confirmed by analysis of the E sigma F-dependent transcription in vivo and in vitro of mutant ssp genes.
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PMID:Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E sigma F: identification of features of good E sigma F-dependent promoters. 174 43

Transcription of the rplKAJLrpoBC ribosomal protein (rpl) RNA polymerase (rpo) gene cluster is governed by a complex set of signals. To dissect the transcription units active in vivo and to quantify the relative contribution of each, an extensive array of rplKAJLrpoB/lacZ gene fusions were constructed on lambda phage derivatives and introduced in single copy into the chromosomes of lac- cells. Measurements of beta-galactosidase production from fusions containing wild-type and/or mutagenized rplrpo DNA fragments permitted the establishment of high-resolution transcription profiles of the gene cluster. The results show that transcription initiated at the upstream rplKp promoter (located just before rplK) does not terminate before the rplJp promoter (located upstream from rplJ), but instead reads through into the distal genes. In addition, rplJp continues to function efficiently in the presence of readthrough transcription from rplKp. As a result the rplJL genes are transcribed at almost twice the frequency of the upstream rplKA genes. However, the transcription of rpoB, which is situated downstream from the previously identified attenuator (rpoBa), is only marginally increased (20%) when both promoters are present. This suggests that although both transcription units overlap, transcriptional termination at rpoBa is modulated in response to the frequency of initiation from both promoters.
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PMID:In vivo analysis of overlapping transcription units in the rplKAJLrpoBC ribosomal protein-RNA polymerase gene cluster of Escherichia coli. 182 52

The tellurite resistance (Ter) determinant of the IncP alpha plasmid RK2Ter, a variant of RK2 (also called RP4), is located between the kilA and korA genes involved in plasmid replication control. Transcriptional and translational fusions were constructed between the gene for beta-galactosidase and the kilA and Ter genes by using the transpositional phage mini-Mu. These fusions indicated that the Ter genes are transcribed in the same direction as kilA and that transcription and translation of the cloned kilA gene are occurring and may not be lethal to the bacterial cell even in the absence of korA. The nucleotide sequence of this region was determined, and three open reading frames (ORFs) were identified. The first ORF codes for KilA, a 28-kDa hydrophilic protein. The second ORF, telA, codes for a hydrophilic protein of 42 kDa. The third ORF, telB, codes for a hydrophobic protein of 32 kDa. This protein appears to be located in the inner membrane of the bacterial cell, since fusions of TelB to alkaline phosphatase were obtained by using TnphoA. All three proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after overproduction using the T7 RNA polymerase/promoter system. The same three proteins were produced when Tes and Ter derivatives of RP4 were expressed in an in vitro transcription-translation system. A single Ser-to-Cys missense mutation in telB was found to be responsible for mutation of RK2 to Ter.
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PMID:Transcriptional analysis, translational analysis, and sequence of the kilA-tellurite resistance region of plasmid RK2Ter. 184 56

CytA, a 27-kDa cytolytic crystal protein of Bacillus thuringiensis subsp. israelensis, is produced only at very low levels by recombinant Escherichia coli cells unless a 20-kDa B. thuringiensis subsp. israelensis protein is also present (K. M. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987; L. F. Adams, J. E. Visick, and H. R. Whiteley, J. Bacteriol. 171:521-530, 1989). However, the data reported here demonstrate that the 20-kDa protein is not required for high-level CytA production in E. coli strains carrying mutations in rpoH, groEL, or dnaK, all of which affect the proteolytic ability of the cells. The 20-kDa protein also increases the amount of CryIVD (another B. thuringiensis subsp. israelensis crystal protein) and LacZX90 (a mutant of beta-galactosidase) made by E. coli. The latter phenomenon is attributable to an increase in the half-life of LacZX90, suggesting that the 20-kDa protein may stabilize this protein. The effect of the 20-kDa protein was also examined in vitro and in a T7 RNA polymerase expression system, and the possible significance of these results for the timing of proteolysis and of 20-kDa protein activity is discussed. Finally, the ability of a single antibody to coimmunoprecipitate CytA and the 20-kDa protein from E. coli extracts provides evidence for a protein-protein interaction that may be related to the mechanism of action of the 20-kDa protein.
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PMID:Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli. 190 Feb 80

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.
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PMID:In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator. 190 59

When Escherichia coli cells are transferred from 30 degrees C to 42 degrees C, transcription from specific promoters recognized by RNA polymerase containing sigma 32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of sigma 32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-lacZ gene fusion. Synthesis of a sigma 32-beta-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation--namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.
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PMID:Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli. 196 16


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