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 portion of the rrnB operon coding for 16 S RNA was modified to permit efficient in vitro transcription by T7 RNA polymerase of full-length, correctly terminated, biologically active 16 S RNA (W. Krzyzosiak et al., 1987, Biochemistry 26, 2353-2364). The 5'-end of the gene was fused to the class III T7 promoter and the 3'-end was modified so that cleavage with MstII would generate correctly terminated RNA upon runoff transcription. The modified gene was placed in pUC19 by a four-way ligation reaction involving linearized pUC19, a 1490-bp fragment of 16 S rDNA, and two synthetic oligodeoxynucleotides. Because of the cohesive end design, phosphorylation of the synthetic oligomers was not necessary. Single and tandem cassette insertions were used to generate single base changes in the C-1400 region of 16 S RNA. Three examples are described. This method is generally applicable to the 16 S RNA molecule as suitable singlecleavage restriction sites allow all regions to be mutated by this approach.
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PMID:An efficiently mutagenizable recombinant plasmid for in vitro transcription of the Escherichia coli 16 S RNA gene. 307 Nov 83

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

Plasmid vectors are described that allow cloning of target DNAs at sites where they will be minimally transcribed by Escherichia coli RNA polymerase but selectively and actively transcribed by T7 RNA polymerase, in vitro or in E. coli cells. Transcription is controlled by the strong phi 10 promoter for T7 RNA polymerase, and in some cases by the T phi transcription terminator. The RNA produced can have as few as two foreign nucleotides ahead of the target sequence or can be cut by RNase III at the end of the target sequence. Target mRNAs can be translated from their own start signals or can be placed under control of start signals for the major capsid protein of T7, with the target coding sequence fused at the start codon or after the 2nd, 11th or 260th codon for the T7 protein. The controlling elements are contained on small DNA fragments that can easily be removed and used to create new expression vectors.
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PMID:Vectors for selective expression of cloned DNAs by T7 RNA polymerase. 331 56

Purified Escherichia coli RNA polymerase containing the heat shock regulatory protein sigma 32 (gprpoH) was used to inject BALB/c mice, and the spleen cells of an immunized mouse were fused to NS1 cells. Three stable cell lines were isolated which produced monoclonal antibodies to sigma 32. The antibodies varied in their ability to bind sigma 32 which was bound to core polymerase. Each of the antibodies was found to inhibit transcription from the rpoD heat shock promoter to a different extent. Extensive homology at the protein level has been observed between various sigma factors. The monoclonal antibodies to sigma 32 were tested for the ability to cross-react with sigma 70 on Western blots. None of the antibodies reacted with electroblotted sigma 70. The sigma 32 content was examined in total cell extracts during heat shock Levels of sigma 32 were found to increase approximately 4-fold over pre-heat shock levels at five min after temperature upshift. These levels remained slightly elevated above pre-heat shock levels at 10 and 15 min after temperature upshift.
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PMID:Studies of the role of the Escherichia coli heat shock regulatory protein sigma 32 by the use of monoclonal antibodies. 355 98

To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.
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PMID:Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. 368 96

Using in vitro techniques we have fused upstream sequences from the malPp promoter (normally activated by the MalT protein) to downstream sequences from the lacZp promoter (normally repressed by the LacI protein). Several hybrid promoters were thus obtained, which were controlled by the MalT protein, but were poorly active. More efficient promoters were then isolated using in vivo selection. Three main conclusions could be derived from the analysis of all of these hybrid promoters. Firstly, the MalT protein seems able to force RNA polymerase to start transcription at any DNA sequence, albeit with a low efficiency. Secondly, the strength of the hybrid promoters is considerably increased if a Pribnow Box is positioned at a precise location with respect to the MalT binding site. Thirdly, the presence of the lac operator, even when properly positioned with respect to the transcription startpoint, does not suffice to permit full repression by the lacI product.
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PMID:The mac promoters: functional hybrid promoters activated by the malT product and repressed by the lacI product. 388 38

In prokaryotic organisms, the control of gene expression is mediated by regulatory proteins that activate or repress transcription. However, the molecular mechanisms of positive and negative control are different. In terms of negative control, repressor proteins bind to sites located within the promoter region and as a consequence sterically interfere with functional binding by RNA polymerase. Here, I examine the properties of a regulatory sequence that specifies catabolite (glucose) repression in the yeast Saccharomyces cerevisiae. Specifically, a DNA segment containing this regulatory site was fused upstream of the intact his3 promoter region and structural gene at several locations. Normally, his3 expression in these derivatives occurs at a basal level which can be induced by conditions of amino-acid starvation. However, in glucose medium, the catabolite regulatory sequence overrides the normal his3 promoter elements and reduces transcription both in normal and starvation conditions. The implication from these results is that in contrast to catabolite repression in Escherichia coli, which is mediated by catabolite-activating protein (CAP), catabolite repression in yeast occurs by a negative control mechanism involving a putative repressor protein. The observation that this regulatory site exerts its repressing effects even when located upstream of an intact promoter region suggests that repression in yeast is not mediated by steric interference between regulatory proteins and the transcriptional apparatus.
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PMID:Negative control at a distance mediates catabolite repression in yeast. 390 16

A Sau3A fragment containing most of the repA gene of plasmid R1 has been cloned in the BamH1 site of the expression vector pAS1. One of the recombinants, pSO1, codes for a fused RepA' protein which is efficiently synthesized both in vivo and in vitro from transcriptional and translational signals of the vector. It is shown that following pSO1 promoted accumulation of RepA' in cell-free extracts of E. coli, in vitro replication of the R1 miniplasmid pKN182 can initiate and proceed uncoupled from further protein synthesis. Using this uncoupled system and also a M13mp9 based ori-R1 recombinant, pRD95, it is also shown that RepA' acts at the origin region of R1 to promote initiation of replication that is independent on transcription by host RNA polymerase. This is indicated by the insensitivity of pRD95 and pKN182 replication to rifampicin as well as to RNA polymerase antibodies. The properties of the uncoupled in vitro replication system are further described.
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PMID:Initiation of plasmid R1 replication in vitro is independent of transcription by host RNA polymerase. 608 75

We have partially characterized a DNA fragment encoding glutamine synthetase in Salmonella typhimurium. Restriction mapping and RNA polymerase binding studies identified two regions within the fragment which exhibit promoter activity when fused to lacZ in pMC1403, a plasmid used to detect transcriptional and translational control signals. DNA sequence analysis revealed that one region encodes amino acids corresponding to the amino terminus of the glutamine synthetase protein. The second region codes for the amino acids corresponding to the carboxy terminus of glutamine synthetase followed by a 330-nucleotide sequence containing an ideal Pribnow heptamer and a possible translation initiation signal. The location of this region is analogous to the position of the beginning of the glnL gene identified in Escherichia coli, and it is likely that the Pribnow heptamer is the RNA polymerase binding site for the glnL gene.
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PMID:Nucleotide sequence of the control regions for the glnA and glnL genes of Salmonella typhimurium. 613 17

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.
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PMID:Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid. 618 Feb 95


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