R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.

R环是当RNA入侵双链DNA与其模板序列配对时形成的核酸杂合体。尽管它们与越来越多的基因调控过程有关，但其机制起源仍不清楚。我们在这里报告在单分子分辨率的共转录R环形成的实时观察，并提出了其形成的机制。我们表明，细菌的Mfd蛋白可以同时与延长RNA聚合酶和上游DNA相互作用，将两者拴在一起并将DNA分成不同的超螺旋结构域。在Mfd和RNA聚合酶之间形成高度负超螺旋结构域，而在RNA聚合酶前面和Mfd后面出现补偿性正超螺旋。新生的RNA进入负超螺旋结构域，并形成一个稳定的R环，可以驱动诱变。从理论上讲，该机制可使任何同时结合主动移位的RNA聚合酶和上游DNA的蛋白质刺激R环的形成。