Volume 272, Number 19,
Issue of May 9, 1997
pp. 12816-12823
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA Topoisomerases Regulate R-loop Formation during Transcription
of the rrnB Operon in Escherichia coli
(Received for publication, December 18, 1996)
Eric
Massé
,
Pauline
Phoenix
and
Marc
Drolet
From the Département de Microbiologie et immunologie,
Université de Montréal, C.P. 6128, Succursale Centre-Ville,
Montréal, Québec H3C 3J7, Canada
Recent in vivo and in
vitro studies have suggested an important role for DNA
topoisomerases in regulating R-loop formation during transcription in
Escherichia coli. In the present report we present genetic
and biochemical evidence strongly suggesting that R-loop formation can
occur during transcription of a portion of the rrnB operon
and that it is regulated by DNA topoisomerase activity. We found that a
multicopy plasmid (pBR322) carrying an heavily transcribed portion of
the rrnB operon cannot be transformed in topA
mutants unless RNase H is overproduced. Transcription of the 567-base
pair HindIII fragment from the rrnB operon
allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular
level of RNase H activity. When DNA gyrase is sufficiently active,
hypernegatively supercoiled plasmid DNA is produced if the same DNA
fragment is transcribed in a topA mutant. The formation of
such topoisomers most likely reflect the presence of extensive R-loops
since it is sensitive to the intracellular level of RNase H activity.
Finally, the formation of R-looped plasmid DNAs in an in
vitro transcription system using phage RNA polymerases is also
detected when the 567-base pair HindIII fragment is
transcribed on a negatively supercoiled DNA template.
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