Escherichia coli DNA topoisomerase I inhibits R-loop formation by relaxing transcription-induced negative supercoiling.
It has recently been shown that RNase H overproduction can partially compensate for the growth defect due to the absence of DNA topoisomerase I in Escherichia coli (Drolet, M., Phoenix, P., Menzel, R., Massé, E., Liu, L. F., and Crouch, R. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3526-3530). This result has suggested a model in which inhibitory R-loops occur during transcription in topA mutants. Results presented in this report further support this notion and demonstrate that transcription-induced supercoiling is involved in R-loop formation. First, we show that stable R-loop formation during in vitro transcription with E. coli RNA polymerase only occurs in the presence of DNA gyrase. Second, extensive R-loop formation in vivo, revealed by the production of RNase H-sensitive hypernegatively supercoiled plasmid DNAs, is observed under conditions where topA mutants fail to grow. Furthermore, we have demonstrated that the coupling of transcription and translation in bacteria is an efficient way of preventing R-loop formation.
Massé
E
E
Département de Microbiologie et immunologie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7.
Drolet
M
M
D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016914:Ribonuclease H / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D000198:Spectinomycin / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics; D014158:Transcription, Genetic
D017920:Antiporters; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011500:Protein Synthesis Inhibitors; C079051:tetA protein, Bacteria; D000198:Spectinomycin; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II