The bacterial transcription terminator, Rho, functions as an RNA:DNA hybrid (RDH) helicase <i>in vivo</i>

Ribonuclease HI (rnhA) removes the deleterious RNA:DNA hybrids (RDHs) by cleaving its RNA component. The bacterial transcription terminator Rho is an RNA-dependent 5' → 3' helicase capable of unwinding RDH formed on a single-stranded RNA in vitro. We hypothesize that Rho might be directly involved in RDH removal in vivo. Here, we demonstrate that Rho primary RNA-binding site (PBS) mutants defective in RNA binding and helicase activity are synthetically lethal specifically when RNase HI is absent. This lethality was not observed in the absence of RNase HII (rnhB) alone. Rho-PBS mutants in an rnhA- strain exhibited increased plasmid-concatemer and plasmid copy number, altered cell morphology, and were highly susceptible to DNA-damaging agents. These Rho mutants increased the accumulation of RDHs in vivo, suggesting defects in the RDH removal process. Rho was colocalized to RDHs in vivo when RNase HI was absent. Certain catalytically inactive mutants of RNase H that bind to the RDH blocked the entry of Rho to the RDH, inducing cell death, indicating the role of Rho in the removal of deleterious RDHs in the absence of RNase HI. Under in vitro conditions, Rho was capable of binding to the RDHs and unwinding them in a rut-site-dependent manner. Therefore, we concluded that in the absence of RNase HI, Rho, by its RNA-dependent helicase activity, is capable of unwinding RDHs in a rut-site-dependent manner. These results establish the non-transcription terminator role of Rho and its functional synergy with RNase HI in vivo.