Abstract
RNA polymerase (RNAP) encounters various roadblocks during transcription. Given that these obstacles can change the dynamics of RNAP movement, they are likely to influence transcription either directly or through RNAP associated factors. One such factor is Mfd; a highly conserved DNA translocase that is thought to primarily function in repair of DNA lesions that have stalled RNAP. However, the interaction between Mfd and RNAP may also be important for transcription regulation at generally hard-to-transcribe regions where RNAP frequently stalls in living cells, even in the absence of DNA lesions. If so, then Mfd may function as a critical RNAP co-factor and a transcription regulator, at least for some genes. This model has not been directly tested.
Here, we assessed the function of Mfd in vivo and determined its impact on RNAP association and transcription regulation. We performed genome-wide studies, and identified chromosomal loci bound by Mfd. We found many genomic regions where Mfd modulates RNAP association and represses transcription. Additionally, we found that almost all loci where Mfd associates and regulates transcription contain highly structured regulatory RNAs. The RNAs in these regions regulate a myriad of biological processes, ranging from metabolism, to tRNA regulation, to toxin-antitoxin functions. We found that transcription regulation by Mfd, at least at the toxin-antitoxin loci, is essential for cell survival. Based on these data, we propose that Mfd is a critical RNAP co-factor that is essential for transcription regulation at difficult-to-transcribe regions, especially those that express structured regulatory RNAs.
Significance The Mfd translocase recognizes stalled RNAPs. This recognition is generally thought to facilitate transcription-coupled DNA repair, based largely on data from biochemical studies. Little is known about Mfd’s function in living cells, especially in the absence of exogenous DNA damage. Our data show that Mfd is a critical RNAP co-factor that modulates RNAP association and regulates transcription at various loci, especially those containing highly structured, regulatory RNAs. This work improves our understanding of Mfd’s function in living cells and assigns a new function to Mfd as a regulator of transcription at hard-to-transcribe regions where maintaining transcriptional equilibrium (e.g. at toxin-antitoxin loci) is essential for viability. Altogether, this work also expands our understanding of how transcription is regulated at difficult-to-transcribe loci.
Competing Interest Statement
The authors have declared no competing interest.