Mfd regulates RNA polymerase association with hard-to-transcribe regions in vivo, especially those with structured RNAs

Abstract

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.

Mfd Affects Global Transcription and the Physiology of Stressed Bacillus subtilis Cells

“…Our lab demonstrated a role for Mfd in the expression of amino acid biosynthesis genes and protection from oxidative stress (Pybus et al, 2010;Martin et al, 2011Martin et al, , 2019. Of note, a recent report proposed that Mfd functions at hard-to-transcribe genes and affected gene expression and survival associated with toxin-antitoxin gene modules in B. subtilis (Ragheb et al, 2021).…”

“…For example, tolerance to diamide in the sodA and mfd sodA mutants was abrogated but overexpression of mfd rescued it, which suggests an Mfddependent effect. Direct effects on genes can be identified by combining transcriptome experiments and assays that test Mfd interactions with the transcription machinery (NETSeq or ChIPSeq) (Ragheb et al, 2021). Similar assays were used recently to characterize transcriptional pauses as affected by NusG (Yakhnin et al, 2020).…”

“…A recent genome association report showed that expression of almost 380 genes were affected by Mfd during exponential growth; genes that were directly affected by this factor encode toxin-antitoxin modules (Ragheb et al, 2021). Further, those studies showed that overexpression of the txpA, bsrH, genes in Mfd − cells decreased cell survival compared to the parent strain and led to propose a model in which Mfd directly interacts with RNAP to modulate transcription of regions with structured RNAs (Ragheb et al, 2021). Interestingly, our transcriptome assays were conducted in stationary-phase cells and showed no changes in expression of txpA and bsrH genes in untreated cells.…”

The Transcription-Repair Coupling Factor Mfd Prevents and Promotes Mutagenesis in a Context-Dependent Manner

“…Mfd recognizes stalled RNAP and displaces it from the damage site while concomitantly recruiting UvrA 2 UvrB ( Selby and Sancar, 1993a ). Even though the Mfd protein has been extensively studied for nearly three decades since it was cloned ( Selby and Sancar, 1993a ) and characterized ( Selby and Sancar, 1993b , 1994 , 1995a,b ) by Selby, several recent reports have significantly advanced our understanding of Mfd, including discoveries from whole-genome analyses ( Adebali et al,2017a,b ; Ragheb et al, 2021 ), as well as from structural ( Brugger et al, 2020 ; Kang et al, 2021 ) and single-molecule ( Fan et al, 2016 ; Ho et al, 2018 , 2020 ; Ghodke et al, 2020 ) studies which will be reviewed here.…”

“…A very recent E. coli whole-genome analysis from Houra Merrikh’s lab mapped Mfd-associated genomic loci using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and found a very high correlation ( r = 0.98) between sites bound by Mfd and those bound by RNAP ( Ragheb et al, 2021 ). Interestingly, this study was performed in the absence of exogenous DNA damage, and the Mfd-bound sites correlated ( r = 0.6) with the sites of the E. coli RNA secondary structure as determined by parallel analysis of RNA structure (PARS-seq).…”