Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter.
We have investigated the formation of the first phosphodiester bond by RNA polymerase II in vitro. The template was a cloned DNA bearing the adenovirus 2 major late promoter; transcription factors and RNA polymerase II were provided by a HeLa cell nuclear extract. Dinucleotide primers and single nucleoside triphosphates were used as substrates. We found that accurate initiation does occur when only one phosphodiester bond can be formed; however, all of the resulting dinucleotide-primed trimers are abortively initiated. Synthesis of the trimers by RNA polymerase II requires ATP or dATP and is sensitive to low concentrations of alpha-amanitin. Treatments which abolish the ability of the preinitiation complex to synthesize long RNAs also eliminate the ability to abortively initiate. Abortive initiation proceeds for at least one-half h at 25 degrees C, at which point up to 4 mol of transcript/mol of template have been synthesized. The level of abortive initiation (per template molecule) is not significantly reduced by 0.025% Sarkosyl or by 10-fold dilution of the reaction, consistent with the initiation complex remaining intact during abortive initiation.
Luse
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Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, Ohio 45267-0522.
Jacob
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GA
D000260:Adenoviruses, Human / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D000546:Amanitins / Q000494:pharmacology; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D005814:Genes, Viral; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism
D000546:Amanitins; D014157:Transcription Factors; D004247:DNA; D012319:RNA Polymerase II