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)

2-Chloro-2'-deoxyadenosine (cladribine [CldAdo]) represents one of the most promising therapeutic agents for the treatment of pediatric leukemias and adult hairy cell leukemia. We examined whether CldAdo incorporation into DNA inhibited subsequent transcription in vitro using purified phage RNA polymerases. Control (Ade-containing) and 2-chloroadenine (ClAde)-substituted DNA strands that contained a RNA polymerase promoter sequence were synthesized by a modified asymmetric polymerase chain reaction. Complementary (+) and (-) strands were annealed, incubated with phage RNA polymerase, and analyzed with denaturing PAGE. When ClAde was present in both strands, the yield of full-length transcripts (approximately equal to 100 bases) was reduced by approximately equal to 90% relative to control DNA. Transcription was also reduced to a slightly lesser degree when substitutions occurred in only one of two strands. The observed low transcript levels on ClAde-containing DNA were due in part to the presence of the analogue within the promoter region. With gel shift binding assays, we demonstrated that RNA polymerase did not bind as well to ClAde-containing promoters. Polymerase/DNA complex formation was decreased by approximately equal to 80% compared with that on control unsubstituted promoters. In addition, on binding to the substituted promoter, RNA polymerase had an altered conformation that led to enhanced proteolytic clipping by endoproteinase Glu-C. Transcript sequence analysis indicated that SP6 RNA polymerase read through template ClAde residues with no apparent misincorporation into RNA. Our results provide insight into a novel effect of this nucleoside analogue that may explain its cytotoxicity in nondividing cells.
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PMID:In vitro transcription of DNA containing 2-chloro-2'-deoxyadenosine monophosphate. 747 21

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.
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PMID:Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2). 894 50