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 regulation of gene expression was studied, for the Escherichia coli rpoBC operon, which includes the genes, rpoB and rpoC, for the beta and beta subunits of RNA polymerase, and rplJ and rplL, for the two proteins, L10 and L7/12, of the 50S ribosome. The gene organization agrees well with the accumulated observations indicating the coordinate synthesis of RNA polymerase and ribosomes under various growth conditions for wild-type E. coli cells. On the other hand, the differential regulation of the two essential components observed under restrictive growth conditions, after addition of various drugs or with certain mutants, in particular those carrying mutations in the RNA polymerase genes, was found to take place through two novel regulation systems: The transcriptional termination at an internal attenuation site and the two autogenous and posttranscriptional controls, being specific for the two ribosomal protein genes and the two RNA polymerase subunit genes, respectively. The majority of the transcription initiated from the promoter rpoP beta terminates at an attenuator site between the promoter-proximal rplJL and the promoter-distal rpoBC genes. The frequency of the attenuation seems to control the relative level of RNA polymerase synthesis to that of ribosomes. The expression of rpoBC genes is subject to an autogenous regulation, in which both RNA polymerase holoenzyme and alpha 2 beta complex function as regulatory molecules with repressor activity. The autogenous regulation was found to operate at post-transcriptional step(s), probably at the level of translation. During the study on the regulation of RNA polymerase synthesis, we noticed that the rpoBC operon contained another autogenous regulation circuit, in which the synthesis of L10 and L7/12 was specifically repressed by the L10-L7/12 complex. Molecular mechanisms and physiological meanings of the novel regulations are discussed.
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PMID:Autogenous and post-transcriptional regulation of RNA polymerase synthesis. 700 54

A partial restriction of ribonucleic acid (RNA) polymerase activity has been used to dissociate the coordinate synthesis of ribosomal proteins and subunits of RNA polymerase and to identify transcriptional and post-transcriptional control signals which regulate the expression of these component genes. Within the beta operon [which has the genetic organization: promoter (p beta), rplJ (L10), r;lL (L7/L12), attenuator, rpoB (beta), rpoC (beta'), terminator], the restriction caused a disproportionate increase between proximal and distal gene transcriptions; the transcriptional intensities of the proximal ribosomal protein genes and the distal RNA polymerase genes were elevated about two- and fourfold, respectively. Transcription within the operon containing four ribosomal protein genes and the RNA polymerase alpha gene was also enhanced, whereas transcription within operons containing only ribosomal protein genes was virtually unaffected by the restriction. It was thus concluded that the mechanisms controlling transcription initiation or attenuation or both in operons containing RNA polymerase subunit genes are coupled to the global rate of RNA synthesis. By introducing the composite ColE1 plasmid pJC701 carrying the proximal portion of the L10 operon, including the beta subunit gene, it was possible to achieve a 10- and a 30-fold range in the transcriptional intensities of the genes specifying L10 and L7/L12 and beta, respectively. Under these conditions, the relative synthesis rates of L7/L12 and beta protein varied by less than 2-fold and by about 15-fold, respectively. These observations corroborate the existence of a post-transcriptional mechanism which severely restricts translation of excess L7/L12 and L10 ribosomal protein messenger RNA; this mechanism is probably important in maintaining the balanced synthesis of ribosome components under conditions in which their messenger RNA levels are dissociated. Furthermore, the observed reduction in the translation efficiency of beta subunit messenger RNA may be related to an inhibitory effect caused by accumulation of RNA polymerase assembly intermediates.
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PMID:Transcriptional and post-transcriptional control of ribosomal protein and ribonucleic acid polymerase genes. 701 43

When 3-day-old etiolated soybean seedlings are treated with the synthetic auxin, 2,4-dichlorophenoxyacetic acid, cells of the mature hypocotyl swell and proliferate abnormally. By 48 h after auxin application ribonucleic acid (RNA) polymerase I and II levels increase by about 10-20- and 6-fold, respectively, on a fresh weight tissue basis and about 3-6- and 2-fold, respectively, on a tissue deoxyribonucleic acid (DNA) basis. [35S]Methionine incorporation into RNA polymerase subunits suggests that this increase in levels of RNA polymerases results from de novo synthesis of the enzymes. No alteration in subunit structure or patterns of incorporation of [35S]methionine into RNA polymerase subunits is detected following auxin treatment. No differences in the phosphorylation patterns of RNA polymerase subunits are detected after hormone treatment. These results indicate that although the levels of RNA polymerases I and II may regulate, in part, the rates of transcription during physiological or developmental transitions, alteration or modification of RNA polymerase subunit structure does not appear to be involved in transcriptional regulation in the auxin-induced soybean hypocotyl.
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PMID:Auxin-induced deoxyribonucleic acid dependent ribonucleic acid polymerase activities in mature soybean hypocotyl. 719 81

Ternary complexes of vaccinia virus RNA polymerase containing 3'-OMeGMP-arrested transcripts were purified by native gel electrophoresis. These complexes resumed elongation in situ when gel slices were incubated with magnesium and NTPs. Elongation occurred in the absence of pyrophosphate, suggesting that the blocking 3'-OMeGMP residue was removed via a novel pathway. We show that purified elongation complexes contain an intrinsic nuclease activity that shortens nascent RNA from the 3'-end. RNA cleavage was absolutely dependent on a divalent cation and was stimulated by CTP. The initial 5' cleavage product remained associated with the ternary complex and could be elongated in the presence of NTPs. Multiple stepwise cleavages generated progressively shorter chains. Purified ternary complexes containing 3'-OH-terminated RNAs also displayed nuclease activity. Involvement of the vaccinia RNA polymerase subunit rpo30 in the transcript-shortening reaction is suggested based on sequence similarity of rpo30 to mammalian protein SII (TFIIS), an extrinsic transcription factor required for nascent RNA cleavage by RNA polymerase II (Reines, D. (1991) J. Biol. Chem. 267, 3795-3800).
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PMID:Nascent RNA cleavage by purified ternary complexes of vaccinia RNA polymerase. 767 16

The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.
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PMID:RNA polymerase II subunit RPB9 is required for accurate start site selection. 788 69

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.
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PMID:C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7. 806 49

Control of tuberculosis is threatened by widespread emergence of drug resistance in Mycobacterium tuberculosis. Understanding the molecular basis of resistance might lead to development of novel rapid methods for diagnosing drug resistance. We set out to determine the molecular basis of resistance to rifampicin, a major component of multidrug regimens used for treating tuberculosis. Resistance to rifampicin involves alterations of RNA polymerase. The gene that encodes the RNA polymerase subunit beta (rpoB) was cloned. Sequence information from this gene was used to design primers for direct amplification and sequencing of a 411 bp rpoB fragment from 122 isolates of M tuberculosis. Mutations involving 8 conserved aminoacids were identified in 64 of 66 rifampicin-resistant isolates of diverse geographical origin, but in none of 56 sensitive isolates. All mutations were clustered within a region of 23 aminoacids. Thus, substitution of a limited number of highly conserved aminoacids encoded by the rpoB gene appears to be the molecular mechanism responsible for "single step" high-level resistance to rifampicin in M tuberculosis. This information was used to develop a strategy (polymerase chain reaction-single-strand conformation polymorphism) that allowed efficient detection of all known rifampicin-resistant mutants. These findings provide the basis for rapid detection of rifampicin resistance, a marker of multidrug-resistant tuberculosis.
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PMID:Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. 809 75

Sigma factor (sigma) is a dissociable subunit of bacterial RNA polymerase that determines promoter recognition. It has been proposed that a cluster of highly conserved aromatic amino acids in bacterial sigma factors (region 2.3) defines a melting motif that functions in strand-separation during open complex formation. We demonstrate that many alterations in region 2.3 of the Bacillus subtilis sigma A protein specifically impair open complex formation. The region 2.3 mutations can be grouped in three classes: (1) mutations that do not significantly affect promoter recognition or melting; (2) mutations that lead to cold-sensitive transcription of linear templates; and (3) mutations that lead to little activity on linear templates but retain activity at high temperatures on supercoiled templates. RNA polymerase holoenzymes containing sigma factor melting mutants (classes 2 and 3) form predominantly closed complexes at 40 degrees C and are defective for RNA synthesis when initiation is rate-limiting. The melting defect of these mutant sigma factors is suppressed by template supercoiling, but further enhanced by inclusion of the auxiliary RNA polymerase subunit delta. Consequently, in the presence of the delta polypeptide, the mutant holoenzymes display cold-sensitive transcription on supercoiled templates: conditions which mimic the in vivo situation. A subset of these mutations also affects promoter selectivity, suggesting that region 2.3 may participate in both -10 recognition and DNA melting.
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PMID:A promoter melting region in the primary sigma factor of Bacillus subtilis. Identification of functionally important aromatic amino acids. 810 87

Enzymes and factors, required for in vitro transcription of templates regulated by vaccinia virus intermediate stage promoters, are present in HeLa cells infected with vaccinia virus in the presence of an inhibitor of DNA replication. Previous studies indicated that in vitro transcription could be reconstituted by adding a partially purified transcription factor to the viral RNA polymerase and capping enzyme. By using an independent purification procedure, we isolated two vaccinia virus intermediate were necessary for transcription of several different intermediate stage promoter templates but not for early or late stage promoter templates. VITF-1 was purified to homogeneity, and the sequences of two tryptic peptides were mapped to the fourth open reading frame within the HindIII E fragment (E4L) of the vaccinia virus genome, which had previously been shown to encode an RNA polymerase subunit of 30 kDa (RPO30) with homology to eukaryotic transcription elongation factor SII. Co-chromatography of VITF-1 with the E4L-derived protein was demonstrated using specific antiserum. In addition, transcriptionally active recombinant VITF-1 was made by expressing the E4L open reading frame in Escherichia coli. Thus, E4L encodes a multifunctional protein, serving as a RNA polymerase subunit and a stage-specific transcription factor. The stepwise binding of capping enzyme, VITF-1, and VITF-2 to a DNA/viral RNA polymerase complex was demonstrated.
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PMID:Purification and identification of a vaccinia virus-encoded intermediate stage promoter-specific transcription factor that has homology to eukaryotic transcription factor SII (TFIIS) and an additional role as a viral RNA polymerase subunit. 818 10

To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed.
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PMID:Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene. 836 91


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