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Volume 271, Number 35,
Issue of August 30, 1996
pp. 21549-21558
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Amanitin Greatly Reduces the Rate of Transcription by RNA
Polymerase II Ternary Complexes but Fails to Inhibit Some Transcript
Cleavage Modes
(Received for publication, June 6, 1996)
Michael D.
Rudd
and
Donal S.
Luse
From the Department of Molecular Biology, Cleveland Clinic
Foundation Research Institute, Cleveland, Ohio 44195 and
Department of Molecular Genetics, Biochemistry and
Microbiology, University of Cincinnati College of Medicine, Cincinnati,
Ohio 45267-0524
The toxin -amanitin is frequently employed to
completely block RNA synthesis by RNA polymerase II. However, we find
that polymerase II ternary transcription complexes stalled by the
absence of NTPs resume RNA synthesis when NTPs and amanitin are added.
Chain elongation with amanitin can continue for hours at approximately
1% of the normal rate. Amanitin also greatly slows pyrophosphorolysis
by elongation-competent complexes. Complexes which are arrested (that
is, which have paused in transcription for long periods in the presence
of excess NTPs) are essentially incapable of resuming transcription in
the presence of -amanitin. Complexes traversing sequences that can
provoke arrest are much more likely to stop transcription in the
presence of the toxin. The substitution of IMP for GMP at the 3 end of
the nascent RNA greatly increases the sensitivity of stalled
transcription complexes to amanitin. Neither arrested nor stalled
complexes display detectable SII-mediated transcript cleavage following
amanitin treatment. However, arrested complexes possess a low level,
intrinsic transcript cleavage activity which is completely
amanitin-resistant; furthermore, pyrophosphorolytic transcript cleavage
in arrested complexes is not affected by amanitin.

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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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