Transcription in vitro of adenovirus-2 DNA by RNA polymerases class C purified from uninfected and adenovirus-infected HeLa cells.
DNA-dependent RNA polymerase class C (or III) has been solubilized from either uninfected or adenovirus-2-infected HeLa cells and purified by chromatography on phosphocellulose, DNA-cellulose, CM-Sephadex and DEAE-Sephadex. The last column separated the enzyme into three forms CI, CII and CIII, which were completely free of RNA polymerases class A and B and of DNase and RNase. The total and the relative amount of these different enzyme C forms did not vary whether purified from uninfected or infected cells. Irrespective of the stage of purification, the three enzyme forms transcribed deproteinized adenovirus-2DNA very efficiently. This transcription was highly sensitive to elevated ionic strength (especially in the presence of Mg2+) and was accompanied by continuous reinitiation as shown by adding poly(rI), a potent inhibitor of initiation. In addition heparin-resistant initiation complexes could be formed at elevated temperature. The RNA synthesized in vitro on deproteinized intact adenovirus-2 DNA by the different forms of RNA polymerase class C, has been characterized. Analysis of the transcripts by gel electrophoresis, RNA self-annealing, hybridization to separated adenovirus-2 DNA strands and to restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that the various regions of the adenovirus-2 genome were randomly transcribed. In addition, hybridization of RNA transcripts labelled at their 5' end by either  [gamma32P]ATP or [gamma-32P]GTP indicated that not only elongation but also initiation occurred randomly through the entire adenovirus-2 genome, irrespective of the form of the enzyme and of the origin of the cells (normal or infected). The results are discussed in terms of the components which are possibly involved in specific transcription.
D000260:Adenoviruses, Human / Q000201:enzymology*; D000546:Amanitins / Q000494:pharmacology; D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D009994:Osmolar Concentration; D012320:RNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D000546:Amanitins; D004279:DNA, Viral; D008345:Manganese; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III; D008274:Magnesium