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 f or DNA topoisomerases in regulating R-loop formation during transcript ion in Escherichia coil, In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occ ur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity, We found that a multicopy pl asmid (pBR322) carrying an heavily transcribed portion of the rrnB ope ron cannot be transformed in topA mutants unless RNase H is overproduc ed. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plas mid DNAs from a topA mutant, in a manner that depends on the intracellular lev el of RNase H activity, When DNA gyrase is sufficiently active, hypern egatively super coiled plasmid DNA is produced if the same DNA fragmen t is transcribed in a topA mutant, The formation of such topoisomers m ost likely reflect the presence of extensive R-loops since it is sensi tive to the intracellular level of RNase H activity, Finally, the form ation of R-looped plasmid DNAs in an in vitro transcription system usi ng phage RNA polymerases is also detected when the 567-base pair HindI II fragment is transcribed on a negatively supercoiled DNA template.