Mechanism of transcription modulation by the transcription-repair coupling factor

Abstract

Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

Molecular Mechanisms of Transcription-Coupled Repair

“…Single-molecule studies have confirmed and extended in great detail the results of ensemble biochemical studies with Mfd (4,5,35,37,39,52). Binding of Mfd to stalled RNAP and displacement of RNAP along with the truncated transcript, and importantly, the time it takes for these reactions, have been observed with single-molecule technology.…”

“…Even though TCR was first discovered in mammalian cells, currently E. coli TCR is the bestunderstood TCR system and the only system that has been reconstituted in vitro with defined DNA substrates, RNAP, repair proteins, and the Mfd (TRCF) translocase purified to homogeneity (30,31). Moreover, the results obtained with ensemble biochemical experiments have been confirmed, refined, and extended by X-ray crystallography (32), cryo-electron microscopy (cryo-EM) (33,34), and in vivo and in vitro single-molecule biochemical analyses (35)(36)(37)(38)(39). Here, to study TCR following induction of the lac operon, cells were incubated with or without IPTG before irradiation.…”

“…domain interacting with its N-terminal D1-D2 domains, which are homologous to the UvrAinteracting domain of UvrB (32, 59) (Figure 4a). Recent studies using cryo-EM (Figure 4b), small angle X-ray scattering, and measurements of translocation (33,34,39,60,61) have provided insight into how Mfd undergoes multiple conformational changes when it interacts with and displaces stalled RNAP while recruiting UvrA 2 UvrB to the site of DNA damage: Domain D4, which contains the RNAP-interaction domain (RID), interacts with the β subunit of RNAP, and although initial binding of the RID to the RNAP does not require conformational changes, ATP hydrolysis is required for Mfd to form a stable complex with RNAP, and this promotes remodeling of Mfd from its repressed conformation to expose the UvrA-interacting surface in D1-D2 and enables the Mfd translocation module (domains D5 and D6) to bind to the upstream DNA duplex. Domains D5-D6 of Mfd are homologous to the RecG bacterial motor protein that couples ATP hydrolysis to dsDNA translocation.…”

Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines

“…Another well-known protein roadblock is a stalled transcription complex. Stalled RNA polymerases (RNAP) remain stably bound to their DNA template , and can therefore inhibit DNA replication. Nuclease-dead Cas9 (dCas9) is a good mimic this type of block, since it is also a nucleoprotein complex that stably binds to DNA.…”