In vitro transcription initiation by purified RNA polymerase II within the adenovirus 2 major late promoter region.
We have mapped a major initiation site of purified calf thymus RNA polymerase II in the cloned adenovirus 2 major late promoter. The specificity of this initiation site has been determined by "run-off" transcriptional analysis and by RNase T1 analysis which employs single-stranded M13 phage DNA containing the Adenovirus 2 major late promoter as probe. The TATAAA region which is used as the start site by the purified RNA polymerase II for in vitro transcription is 30 base pairs upstream from the adenovirus major late in vivo start site. The exact sequence also exists at two sites within the pBR322 plasmid but initiation does not occur at either of these sites. This indicates that the purified enzyme is not just recognizing AT-rich regions but that it is recognizing both the TATA box and its surrounding sequences. The purified RNA polymerase II transcriptional initiation site is used when transcription was carried out on either a superhelical (FI) or linear (FIII) DNA template. Selective initiation of transcription on FI DNA required (NH4)2SO4 concentrations which ranged from 90 to 150 mM. In contrast, selective initiation of transcription on FIII DNA was observed at (NH4)2SO4 concentrations that ranged from 30 to 120 mM.
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009876:Operon; D010957:Plasmids; D012319:RNA Polymerase II / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology*; D014158:Transcription, Genetic
D012319:RNA Polymerase II; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015719:Single-Strand Specific DNA and RNA Endonucleases