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 DNA-dependent RNA polymerase from Pseudomonas BAL-31, the host for bacteriophage PM2, has been purified 154-fold using differential centrifugation, chromatography on DEAE-cellulose, ammonium sulfate precipitation, and sucrose gradient centrifugations at low and high ionic strength. The resulting enzyme is free of enzyme activities which could interfere with transcription studies and is greater than 85% pure as judged by polyacrylamide gel electrophoresis. Like other bacterial RNA polymerases, its subunit structure is beta'beta sigma alpha2. From gel electrophoresis the beta', beta, and alpha subunits have approximately the same molecular weights as those from Escherichia coli, whereas the sigma subunit is 5% larger (89,000 vs. 85,000). A summation of the subunits yields a molecular weight of 485,000 for the holoenzyme. Like other bacterial RNA polymerases, it sediments as a monomer (15 S) at low ionic strength (0.065) and as a dimer (22 S) at high ionic strength (0.75). Its activity is stimulated three-fold by monovalent cations (K+,NH4+, NA+) with additional stimulation provided by divalent cations (Mg2plus, Mn2plus). The transcription of phage PM2 form I (supercoiled) DNA has an ionic strength optimum of 0.26 for continuous long-term synthesis, and over an ionic strength range of 0.09-0.46 "plateau-type" kinetics are not observed. The sigma subunit is required for optimal PM2 transcription. The enzyme is sensitive to the same inhibitors of transcription as the RNA polymerase from E. coli, it has a temperature optimum of 28 degrees, and it is 50% inactivated by heating 10 min at 41 degrees. It has template preference similar to E. coli polymerase and shows little preference for homologous templates. With various DNAs the order of template activities is T7 greater than PM2 I congruent to T4 greater than PM2 II (relaxed circular form) greater than lambda-c greater than calf thymus greater than BAL-31 DNA. Phage PM2 form I DNA is transcribed at a twofold greater rate than PM2 form II DNA by this enzyme.
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PMID:DNA-dependent RNA polymerase from Pseudomonas BAL-31. I. Purification and properties of the enzyme. 112 Jan 4

Transcription of the supercoiled form (I) and the relaxed circular form (II) of bacteriophage PM2 DNA was studied utilizing the DNA-dependent RNA polymerase from its host, Pseudomonas BAL-31. Transcription of both templates is continuous for up to 2 hr, but proceeds at a two-fold higher rate on I than on II. This difference is mainly due to a 2.2-fold higher rate of chain initiation on I. When rifampicin (Rif) is added ater 10 min of synthesis, (1) transcription of II ceases by 30 min with a maximum product length of 7000 nucleotides (number average) being produced; (2) transcription of I continues with little rate reduction and with the product reaching 16,000 nucleotides (number average) by 2 hr. Sucrose gradient analysis shows that the product of II achieves maximum size 20 min after Rif addition and sediments in three peaks of 24, 33, and 39 S (approximately one-third, two-thirds, and one genome lengths). The product of I has a heterogeneous distribution and grows continuously with a large fraction reacting greater than 3 genome lengths by 90 min. The same differences in synthesis kinetics, Rif inhibition, and product size distribution are observed when I and II are transcribed by Escherichia coli RNA polymerase. These experiments show that (i) PM2 form I DNA is transcribed mainly by a process of continuous chain elongation, with little chain termination occurring; (ii) PM2 form II is transcribed by a process of continuous chain initiation, elongation, and termination of yield discrete products. Thus, the tertiary structure of circular DNA influences chain termination by RNA polymerase.
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PMID:DNA-dependent RNA polymerase from Pseudomonas BAL-31. II. Transcription of the allomorphic forms of bacteriophage PM2 DNA. 112 Jan 5

A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the minigene plasmid was supercoiled.
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PMID:In vitro analysis of the pea chloroplast 16S rRNA gene promoter. 258 29

Specific binding sites of BAL 31 RNA polymerase on PM2 DNA have been mapped by protection against HincII and HindIII cleavage and by observation of enzyme-DNA complexes by electron microscopy. Nine specific binding sites were observed at map units 0.19, 0.20, 0.28, 0.54, 0.63, 0.65, 0.71, 0.72, and 0.75 by the first method. All these sites were confirmed by electron microscopy which, in addition, revealed another site at 0.05 map unit. Published nucleotide sequences of the region surrounding sites at 0.71 and 0.75 map units show the presence of consensus sequences for procaryotic promoters.
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PMID:Binding of BAL 31 RNA polymerase to PM2 DNA as determined by electron microscopy and protection against restriction endonuclease cleavage. 284 87

The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene.
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PMID:Molecular cloning and characterization of the purE operon of Escherichia coli. 302 90

We used BAL-31 nuclease to delete sequences that surround the transcription initiation site of Drosophila ribosomal DNA. A series of deletions was used as templates for in vitro transcription in a Drosophila cell-free system to identify sequences that influence the activity of RNA polymerase I. Sequences that lie upstream of the site of transcription initiation (nucleotide + 1) affect ribosomal RNA synthesis. We show that the major promoter of polymerase I involves the sequence -43 to -27 and that the region between nucleotides -18 and +20 contains sequences capable of sustaining a low level of accurate transcription.
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PMID:Localization of DNA sequences promoting RNA polymerase I activity in Drosophila. 640 9

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.
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PMID:In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA. 1692 57