DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli.
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.
Massé
E
E
Département de Microbiologie et immunologie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada.
Phoenix
P
P
Drolet
M
M
D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D004926:Escherichia coli; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D014158:Transcription, Genetic; D012407:rRNA Operon
D004278:DNA, Superhelical; D015247:Deoxyribonuclease HindIII; D016914:Ribonuclease H; D004264:DNA Topoisomerases, Type I