RNA polymerase (RNAP) encounters various roadblocks during transcription. These obstacles can impede RNAP movement and influence transcription, ultimately necessitating the activity of RNAP-associated factors. One such factor is the bacterial protein Mfd, a highly conserved DNA translocase and evolvability factor that interacts with RNAP. Although Mfd is thought to function primarily in the repair of DNA lesions that stall RNAP, increasing evidence suggests that it may also be important for transcription regulation. However, this is yet to be fully characterized. To shed light on Mfd’s in vivo functions, we identified the chromosomal regions where it associates. We analyzed Mfd’s impact on RNAP association and transcription regulation genome-wide. We found that Mfd represses RNAP association at many chromosomal regions. We found that these regions show increased RNAP pausing, suggesting that they are hard to transcribe. Interestingly, we noticed that the majority of the regions where Mfd regulates transcription contain highly structured regulatory RNAs. The RNAs identified regulate a myriad of biological processes, ranging from metabolism to transfer RNA regulation to toxin–antitoxin (TA) functions. We found that cells lacking Mfd are highly sensitive to toxin overexpression. Finally, we found that Mfd promotes mutagenesis in at least one toxin gene, suggesting that its function in regulating transcription may promote evolution of certain TA systems and other regions containing strong RNA secondary structures. We conclude that Mfd is an RNAP cofactor that is important, and at times critical, for transcription regulation at hard-to-transcribe regions, especially those that express structured regulatory RNAs.

39RNA polymerase (RNAP) encounters various roadblocks during transcription. Given that 40 these obstacles can change the dynamics of RNAP movement, they are likely to influence 41 transcription either directly or through RNAP associated factors. One such factor is Mfd; a highly 42 conserved DNA translocase that is thought to primarily function in repair of DNA lesions that 43 have stalled RNAP. However, the interaction between Mfd and RNAP may also be important for 44 transcription regulation at generally hard-to-transcribe regions where RNAP frequently stalls in 45 living cells, even in the absence of DNA lesions. If so, then Mfd may function as a critical RNAP 46 co-factor and a transcription regulator, at least for some genes. This model has not been directly 47 tested. 48Here, we assessed the function of Mfd in vivo and determined its impact on RNAP 49 association and transcription regulation. We performed genome-wide studies, and identified 50 chromosomal loci bound by Mfd. We found many genomic regions where Mfd modulates RNAP 51 association and represses transcription. Additionally, we found that almost all loci where Mfd 52 associates and regulates transcription contain highly structured regulatory RNAs. The RNAs in 53 these regions regulate a myriad of biological processes, ranging from metabolism, to tRNA 54 regulation, to toxin-antitoxin functions. We found that transcription regulation by Mfd, at least at 55 the toxin-antitoxin loci, is essential for cell survival. Based on these data, we propose that Mfd is 56 a critical RNAP co-factor that is essential for transcription regulation at difficult-to-transcribe 57 regions, especially those that express structured regulatory RNAs. 58 59 60 61 62 Significance 65 The Mfd translocase recognizes stalled RNAPs. This recognition is generally thought to facilitate 66 transcription-coupled DNA repair, based largely on data from biochemical studies. Little is 67 known about Mfd's function in living cells, especially in the absence of exogenous DNA 68 damage. Our data show that Mfd is a critical RNAP co-factor that modulates RNAP association 69 and regulates transcription at various loci, especially those containing highly structured, 70 regulatory RNAs. This work improves our understanding of Mfd's function in living cells and 71 assigns a new function to Mfd as a regulator of transcription at hard-to-transcribe regions where 72 maintaining transcriptional equilibrium (e.g. at toxin-antitoxin loci) is essential for viability. 73 Altogether, this work also expands our understanding of how transcription is regulated at 74 difficult-to-transcribe loci. 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91Timely and efficient transcription is a fundamental requirement for maintaining cellular 92 homeostasis. The process of transcription elongation is discontinuous, with RNA polymerase 93 (RNAP) processivity altered by a wide range of obstacles. These obstacles vary in severity, 94 from pause sites that slow the rate of RNAP(1-3) to more severe obstacles, such as protein...

Pathogenic bacteria and their eukaryotic hosts are in a constant arms race. Hosts have numerous defense mechanisms at their disposal that not only challenge the bacterial invaders, but have the potential to disrupt molecular transactions along the bacterial chromosome. However, it is unclear how the host impacts association of proteins with the bacterial chromosome at the molecular level during infection. This is partially due to the lack of a method that could detect these events in pathogens while they are within host cells. We developed and optimized a system capable of mapping and measuring levels of bacterial proteins associated with the chromosome while they are actively infecting the host (referred to as PIC-seq). Here, we focused on the dynamics of RNA polymerase (RNAP) movement and association with the chromosome in the pathogenic bacterium Salmonella enterica as a model system during infection. Using PIC-seq, we found that RNAP association patterns with the chromosome change during infection genome-wide, including at regions that encode for key virulence genes. Importantly, we found that infection of a host significantly increases RNAP backtracking on the bacterial chromosome. RNAP backtracking is the most common form of disruption to RNAP progress on the chromosome. Interestingly, we found that the resolution of backtracked RNAPs via the anti-backtracking factors GreA and GreB is critical for pathogenesis, revealing a new class of virulence genes. Altogether, our results strongly suggest that infection of a host significantly impacts transcription by disrupting RNAP movement on the chromosome within the bacterial pathogen. The increased backtracking events have important implications not only for efficient transcription, but also for mutation rates as stalled RNAPs increase the levels of mutagenesis.