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J. Biol. Chem., Vol. 262, Issue 31, 14990-14997, 11, 1987
DS Luse and GA Jacob
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.
Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter
Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, Ohio 45267-0522.
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