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(Received for publication, December 26, 1995, and in revised form, May 20, 1996)
From the Institut für Physikalische Biologie,
Heinrich-Heine-Universität Düsseldorf,
Universitätsstraße 1, D-40225 Düsseldorf,
Federal Republic of Germany
We have studied the response of the effector
molecule guanosine 3
Volume 271, Number 39,
Issue of September 27, 1996
pp. 23884-23894
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,5-bisdiphosphate (ppGpp) on RNA polymerase
pausing during in vitro transcription elongation. Pausing
was followed during single round extension of stalled ternary complexes
excluding possible ppGpp effects on initiation. The ppGpp dependences
of early pausing sites within different transcription systems
controlled by promoters with known response to enhanced ppGpp levels
in vivo were quantitatively characterized. Transcription of
stable RNAs and mRNA genes were analyzed. In addition, the in
vitro pausing behavior of two promoter variants directing the
same sequence but differing in their in vivo ppGpp
sensitivity were compared. In the presence of ppGpp we noted a slight
general enhancement of specific pauses in all transcription systems.
However, genes known to be under stringent or growth rate control
in vivo revealed a notably stronger pausing enhancement.
The sites of pausing are not changed by the presence of ppGpp but
appear to be sequence-specific. The effect of ppGpp on the extent of
pausing depends on the particular promoter and closely adjacent
sequences that the RNA polymerase has passed during initiation. Pausing
enhancement requires the presence of ppGpp during elongation but not
during initiation. The results underline the importance of pausing for
transcription regulation and offer a plausible explanation for
inhibition of stable RNA expression under conditions of elevated
concentrations of ppGpp.
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