R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low...

We have recently presented evidence that inhibitory R-loops form during transcription in topA null mutants when the nascent RNA anneals with the template DNA strand behind the moving RNA polymerase. This was supported by the results of in vitro transcription assays and by in vivo studies in which R-loop formation was shown to be inhibited by coupled transcription-translation. The results presented here support this model and further demonstrate the link between R-loop formation and growth inhibition of topA null mutants. First, we show that RNase H activity is essential in the absence of DNA topoisomerase I. This was observed even if the growth of the topA null mutant is compensated for by naturally selected mutations, that also reduce global supercoiling below the wild-type level. Second, we show that R-loop-dependent hypernegative supercoiling increases as the temperature decreases and correlates with growth inhibition of topA null mutants. In fact, RNase H overproduction is shown to be detrimental to cell growth at 21 degrees C. Presumably, several mRNAs are being sequestered in R-loops and their degradation by RNase H significantly impedes protein synthesis. We propose that a reduced transcription velocity at low temperatures favors the annealing of the nascent RNA with the template strand behind the moving RNA polymerase, in agreement with the results of previous studies. Finally, based on the currently available data on R-loop formation, we present a model that explains the sensitivity of topA null mutants to various environmental changes that are often accompanied by transient inhibition of translation.