Role of E.coli Transcription-Repair Coupling Factor Mfd in Nun-mediated Transcription Termination

DNA replication fork movement is impeded by collisions with transcription elongation complexes (TEC). We propose that a critical function of transcription termination factors is to prevent TEC from blocking DNA replication and inducing replication fork arrest, one consequence of which is DNA double-strand breaks. We show that inhibition of Rho-dependent transcription termination by bicyclomycin in Escherichia coli induced double-strand breaks. Cells deleted for Rho-cofactors nusA and nusG were hypersensitive to bicyclomycin, and had extensive chromosome fragmentation even in the absence of the drug. An RNA polymerase mutation that destabilizes TEC (rpoB*35) increased bicyclomycin resistance >40-fold. Double-strand break formation depended on DNA replication, and can be explained by replication fork collapse. Deleting recombination genes required for replication fork repair (recB and ruvC) increased sensitivity to bicyclomycin, as did loss of the replication fork reloading helicases rep and priA. We propose that Rho responds to a translocating replisome by releasing obstructing TEC.DNA polymerase/RNA polymerase collisions | pulsed-field gel electrophoresis | SOS D NA replication forks translocate 20 to 30 times faster (∼600 nt/s) than the rate of RNA polymerase (RNAP) elongation (∼20 nt/s) (1-3). This finding raises the possibility of repeated collisions between replication forks and transcription elongation complexes (TEC), even when DNA and RNA synthesis are codirectional. Collisions between replication forks and TEC can generate fork collapse and chromosomal double-strand breaks (DSBs) (4, 5). Mechanisms have evolved, therefore, to reduce collisions and to repair collapsed forks. Fork repair is promoted by multiple pathways that involve a variety of DNA repair functions (6). Recombination can restart replisomes blocked by arrested TEC (7,8). Boubakri et al. (9) have demonstrated that DNA helicases Rep, DinG, and UvrD resolve head-on collisions between TEC in rrn operons and replisomes. A recent report describes the role of transcription elongation factors DksA and GreA in preventing conflicts between replication and transcription (7, 10). In vitro, replication forks that collapse after colliding with a codirectional TEC can restart, using an R-loop (RNA-DNA hybrid) from a transcription bubble as primer (11), although it is not known if this occurs in vivo.Transcription termination isolates transcription units and prevents inappropriate expression of downstream genes. Termination in Escherichia coli is largely mediated by Rho, an RNAdependent ATPase that terminates transcription promoter-distal to unstructured and untranslated RNA (12, 13). The RNA/DNA helicase activity of Rho promotes termination by unwinding RNA/DNA hybrid in the RNAP transcription bubble (14). Inhibition of Rho with bicyclomycin (BCM) or deletion of the Rho accessory factors, NusA and NusG, massively disregulate gene expression in E. coli (15). Surprisingly, efficient transcription termination is only required to suppress expression of cryp...

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“…Mfd also terminates TEC arrested by protein roadblocks or by phage HK022 Nun protein (25,26). As shown in Fig.…”

Section: Resultsmentioning

confidence: 73%

Transcription termination maintains chromosome integrity

Washburn

Gottesman²

2010

Proc. Natl. Acad. Sci. U.S.A.

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108

152

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“…In the case of head-on collisions of RNAP with replication forks (11), as well as transcriptional termination (17) and carbon catabolite repression (16), only the RNAP displacement activity of TRCF is essential, whereas the NER recruitment function is at least dispensable, if not detrimental. Our analysis thus brings insight into a general mechanism for UvrA-binding inhibition in TRCFs and suggests that TRCF function relies on a fine temporal and context-dependent tuning of its various activities.…”

Section: Discussionmentioning

confidence: 99%

“…This initiates a cascade of events resulting in lesion excision and gap filling (4,10). TRCF also has roles beyond TCR-in the rescue of replication forks stalled by head-on collisions with RNAPs (11), in the development of antibiotic resistance (12,13), recombination (14,15), and transcriptional regulation (16,17).…”

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confidence: 99%

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Deaconescu

Sevostyanova²,

Artsimovitch³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

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Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.cysteine cross-linking | transcription | ATPase stimulation | UvrB R NA polymerase (RNAP) stalled at DNA lesions on the transcribed strand elicits a preferential pathway for nucleotide excision repair (NER) called transcription-coupled repair (TCR), which is present in Bacteria and Eukarya (1). Bacterial transcription-repair coupling factor (TRCF; also known as Mfd) orchestrates this process by specific recognition of the transcription and NER assemblies, which reflects its twofold role. First, TRCF relieves transcription-dependent NER inhibition due to occlusion of the DNA lesion by RNAP (2). TRCF, an SF2 ATPase with dsDNA translocase but no helicase activity (3), approaches the stalled RNAP from behind and induces its forward translocation by stepping on dsDNA using ATP hydrolysis (4, 5). The consequent collapse of the upstream end of the transcription bubble leads to massive destabilization of the otherwise stable ternary elongation complex (TEC) and transcription termination (4-7). Rho, the only other known bacterial enzymatic terminator, induces termination by a similar forward-translocation mechanism, but translocates along the nascent RNA (8). Second, TRCF recruits the Uvr(A)BC endonuclease to the unmasked lesion by binding to UvrA (4, 9). This initiates a cascade of events resulting in lesion excision and gap filling (4, 10). TRCF also has roles beyond TCR-in the rescue of replication forks stalled by head-on collisions with RNAPs (11), in the development of antibiotic resistance (12, 13), recombination (14, 15), and transcriptional regulation (16, 17).The crystal structure of apo TRCF (18) revealed a multimodular enzyme with eight domains connected by flexible linkers (Fig. 1A), an architecture that appears primed for large conformational changes, which are believed to be critical for coupling RNAP recognition to recruitment of NER enzymes. Domains D1 and D2 of TRCF are similar to the NER protein UvrB, which also binds UvrA (18,19), suggesting that these domains serve as a pl...

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“…First, nutL lies only 34 bp from pL, whereas nutR is separated by 227 bp from pR (14). The proximity of nutL to its cognate promoter may block access of host transcription repair-coupling factor to the Nun-arrested TEC, thus prolonging the half-life of the complex (14). Second, the pL operon includes the RNase III-sensitive site, rIII, which lies just distal to nutL ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In vitro, Nun alone blocks elongation of the TEC (11), whereas in vivo, Nun acts in association with four Escherichia coli host factors, NusA, NusB, NusE, and NusG (11)(12)(13). Host transcription repair-coupling factor releases Nun-arrested TEC (14). The penultimate Nun tryptophan residue (Trp 108 ) mediates the interaction of Nun with the template in the TEC, possibly by intercalating into double-stranded DNA template (15).…”

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confidence: 99%

Transcription Termination by Phage HK022 Nun Is Facilitated by COOH-terminal Lysine Residues

Kim

Gottesman

2004

Journal of Biological Chemistry

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The 109-amino acid Nun protein of prophage HK022 excludes superinfecting bacteriophage by blocking transcription elongation on the chromosome. Multiple interactions between Nun and the transcription elongation complex are involved in this reaction. The Nun NH 2 -terminal arginine-rich motif binds BOXB sequence in nascent transcripts, whereas the COOH terminus binds RNA polymerase and contacts DNA template. Nun Trp 108 is required for interaction with DNA and transcription arrest. We analyzed the role of the adjacent Lys 106 and Lys 107 residues in the Nun reaction. Substitution of the lysine residues with arginine (K106R/ K107R) had no effect on transcription arrest in vitro or in vivo. Nun K106A/K107A was partially active, whereas Nun K106D/K107D was defective in vitro and failed to exclude . All mutants bound RNA polymerase and BOXB. In contrast to Nun K106R/K107R and K106A/ K107A, Nun K106D/K107D did not cross-link DNA template. These results suggest that transcription arrest is facilitated by electrostatic interactions between positively charged Nun residues Lys 106 and Lys 107 and negatively charged DNA phosphate groups. These may assist intercalation of Trp 108 into template.

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Role of E.coli Transcription-Repair Coupling Factor Mfd in Nun-mediated Transcription Termination

Cited by 53 publications

References 19 publications

Transcription termination maintains chromosome integrity

Transcription termination maintains chromosome integrity

Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Transcription Termination by Phage HK022 Nun Is Facilitated by COOH-terminal Lysine Residues

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