Animal DNA-dependent RNA polymerases. Partial purification and properties of three classes of RNA polymerases from uninfected and adenovirus-infected HeLa cells.
DNA-dependent RNA polymerase was solubilized from normal and adenovirus-2 infected HeLa cells. Multiple peaks of enzyme activity were separated by DEAE-Sephadex chromatography. In addition to class A and B enzyme activities (respectively insensitive and sensitive to inhibition by 10 nM alpha-amanitin), three peaks of class C enzyme activity were found which are sensitive to inhibition by alpha-amanitin only at much higher concentrations (0.1 mM). Rechromatography of these class C peaks indicates that they are not chromatographic artifacts. Class C enzymes differ from class A and B enzymes by several criteria including inhibition by alpha-amanitin, immunological properties, and the ability to transcribe native calf thymus DNA at high ionic strength. However, the ionic strength optimum and the divalent cation requirements of class C enzymes are not invariant characteristics of the enzymes and are markedly dependent on the nature and the amount of template in the reaction. No differences, either qualitative or quantitative, were found between the multiple enzymes isolated from normal or adenovirus-2 infected cells. All of the partially purified HeLa cell RNA polymerases were able to transcribe an intact double-stranded adenovirus-2 DNA under conditions where no transcription occurred with purified calf thymus AI and B RNA polymerases. Since the multiple enzymes were devoid of endonuclease and exonuclease activities, the ability of the partially purified enzymes to transcribe adenovirus-2 DNA cannot be ascribed to initiation of RNA synthesis at nicks of single-stranded regions of the DNA. No differences in transcriptional ability between corresponding enzyme classes from normal or infected cells, but a comparison of the ability of the various enzyme classes to transcribe intact viral DNA revealed large differences. Although partially purified HeLa class A and B enzymes were able to initiate on the intact viral DNA to a limited extent only, it appears that the class C enzymes transcribe intact duplex DNA much more efficiently than any other class of eukaryotic polymerase yet reported.
D000256:Adenoviridae / Q000201:enzymology*; D000546:Amanitins / Q000494:pharmacology; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009994:Osmolar Concentration; D011233:Precipitin Tests; D012995:Solubility; D013698:Templates, Genetic
D000546:Amanitins; D007527:Isoenzymes; D008345:Manganese; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium