Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism

Abstract

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a "release and catch-up" mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle's resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution.

Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation

“…The PcrA and Mfd translocases physically interact with stalled RNAPs at lesions on the DNA template. PcrA is a pro-backtracking factor by promoting forward RNAP translocation, and Mfd might be an anti-backtracking that dislodges a stalled RNAP, as previously postulated for the isolated protein in vitro (Selby and Sancar, 1993;Ayora et al, 1996;Park et al, 2002;Deaconescu et al, 2006;Epshtein et al, 2014;Sanders et al, 2017;Ho et al, 2018;Le et al, 2018). It is likely that when damaged template bases interfere with RNAP progression, it backtracks and becomes transiently arrested.…”

“…PcrA-like enzymes contribute to release stalled RTCs with the subsequent recruitment of repair factors or to modulate the re-initiation of DNA replication and transcription (Guy et al, 2009 ; Boubakri et al, 2010 ; Merrikh et al, 2015 ). Several B. subtilis translocases, namely the SF1 DNA helicases (as PcrA, HelD, RecD2) and the SF2 enzymes (Mfd, HepA [also termed YqhH] and YwqA) have been shown to interact with the RNAP (Selby and Sancar, 1993 ; Muzzin et al, 1998 ; Sukhodolets et al, 2001 ; Shaw et al, 2008 ; Yawn et al, 2009 ; Wiedermannova et al, 2014 ; Sanders et al, 2017 ; Le et al, 2018 ). The poorly characterized HepA and YwqA enzymes, which belong to the Swi2/Snf2 family of translocases, share ~30% identity to HepA Eco .…”

“…The poorly characterized HepA and YwqA enzymes, which belong to the Swi2/Snf2 family of translocases, share ~30% identity to HepA Eco . These translocases actively process a RNAP backwards as in the case of a RNAP stalled elongation complex (backtracking) or remove RNAP from the DNA template to resolve RTCs ( Table S2 ) (Muzzin et al, 1998 ; Sukhodolets et al, 2001 ; Shaw et al, 2008 ; Yawn et al, 2009 ; Wiedermannova et al, 2014 ; Sanders et al, 2017 ; Le et al, 2018 ). The contribution of these functions to the inviability of PcrA depletion will be tested.…”

The transcription-repair coupling factor Mfd associates with RNA polymerase in the absence of exogenous damage

“…The identity of Mfd intermediates that dissociate with a 30 s lifetime in Δ uvrA cells remains less certain. These could potentially represent abortive intermediates or translocating Mfd following RNAP displacement where subsequent dissociation may occur stochastically 46 or upon encountering roadblocks 42 .…”

Archaeal DNA Repair Mechanisms

“…The eukaryotic TRCF, CSB, also requires DNA sequences upstream of a stalled RNAP. Mfd catches up to backtracked or stalled polymerases by "autonomously" patrolling DNA upstream of TECs [138]. Deletion of Mfd in Bacteria and Eta in Archaea produce a UV sensitivity phenotype, further suggesting they share an analogous role [136,139].…”