Biology subjects

Campbell, E. A.

Publications and source records attributed to Campbell, E. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Predicting the protein interaction landscape of a mycobacterial pathogen

High-dimensional phenotypic screens of bacterial loss-of-function mutant libraries have determined gene-gene connections and specific phenotypes for thousands of bacterial genes in many species, but deciphering the underlying mechanisms remains decidedly low-throughput. Here, we demonstrate the utility of proteome-wide AI-based protein-protein interaction (PPI) predictions for overcoming this gap by using pooled-AlphaFold3 to assess all [~]1.3 million possible pairwise interactions in the proteome of Mycobacterium leprae. We identify [~]2,000 strong and intermediate PPIs that underlie a significant fraction of phenotypes and gene-gene connections observed in large-scale chemical genomics screens from Mycobacterium tuberculosis, Mycobacterium smegmatis, and Corynebacterium glutamicum. This combined approach predicts specific functions for dozens of previously uncharacterized core, conserved, and essential mycobacterial proteins. We highlight new information derived from the study, including insights into mycobacterial envelope assembly, peptidoglycan remodeling, and new modulators of the central dogma enzymes RNA polymerase and DNA gyrase. These data establish combined pooled-AlphaFold3 PPI prediction and high-throughput genomics approach as the gold standard for large-scale characterization of protein function.

systems biology↗

bioRxiv2026

RNA polymerase inhibitors reveal active-site motions essential for the nucleotide-addition cycle

The nucleotide-addition cycle (NAC) of multi-subunit DNA-dependent RNA polymerases (RNAPs) involves coordinated conformational changes in conserved active-site structural elements, including the trigger loop (TL). The TL is open (unfolded) in most RNAP structures but can close (fold) in substrate-bound (post- or pre-translocated) states of the RNAP, promoting catalysis. TL closure has been associated with closure of another conserved structural element, the Rim-Helices/F-loop (RH-FL), but the role of the RH-FL in the NAC is unclear. Antibiotic leads CBR9379 and AAP-SO2 inhibit the Escherichia coli and Mycobacterium tuberculosis RNAPs, respectively, by binding in a pocket formed by the bridge helix and RH-FL. The precise mechanism of action for these inhibitors is yet to be defined. We present cryo-electron microscopy structures showing that both compounds inhibit the RNAP NAC by preventing RH-FL closure, thereby allosterically destabilizing the closed TL. This work reveals a conserved mechanistic principle of RNAP catalysis across all domains of life and provides new insight for antibiotic design.

biochemistry↗

bioRxiv2025

Structural basis for loading of Transcription Repair-Coupling factor Mfd onto stalled elongation complexes

Transcription-coupled repair (TCR) is a nucleotide excision repair sub-pathway that preferentially removes lesions from the DNA template-strand stalling RNA polymerase (RNAP) elongation complexes (ECs). In bacteria, the superfamily 2 Mfd translocase mediates TCR by displacing stalled ECs and recruiting Uvr(A)B. Using cryo-electron microscopy, we previously visualized seven Mfd-EC complexes spanning the ATP-dependent Mfd loading and EC displacement pathway [L1 -> L2(ADP) -> C1(ATP) -> C2(ATP) -> C3(ADP) -> C4(ADP) -> C5(ATP)]. The first intermediate (L1) was poorly resolved (4.1 [A] nominal resolution) due to low particle occupancy. The pathway is characterized by very large Mfd structural transitions, notably the L1 -> L2 transition. Here, we pre-loaded Mfd with ATP in the presence of the{gamma} -phosphate mimic, BeF3-, limiting rounds of ATP hydrolysis. The resulting accumulation of early intermediates allowed us to resolve the L1 intermediate to 3.5 [A] nominal resolution, revealing bound ADP-BeF3-. We also identified a new intermediate between L1 and L2, L1.5, providing further insight into Mfd conformational changes during loading. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/674597v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1472098org.highwire.dtl.DTLVardef@acfca3org.highwire.dtl.DTLVardef@fe4424org.highwire.dtl.DTLVardef@12bc521_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Ceudovitox: a novel pseudotyped-virus screening platform to identify cell entry factors for high consequence infections.

Development of scalable and highly adaptable platforms to characterise high consequence infections are essential for limiting the impact of future viruses with pandemic potential. As part of this, an understanding of the virus-host interactions is vital, with the cell entry mechanism being crucial for the development of therapeutics and vaccines. Current approaches in this field depend on assays with authentic (live) viruses that require high containment facilities. However, these are hindered by high costs, need for highly trained staff, slow processing times and limited scalability. Using pseudotyped viruses (PV) expressing the chikungunya (CHIKV) envelope protein as a proof-of-principle, we developed a novel screening platform, Ceudovitox, to identify cellular factors involved in viral entry. PVs were engineered to express the herpes simplex virus-1 thymidine kinase, which following addition of ganciclovir, can selectively kill infected cells. Then a heterogenous pool of knockout cells were produced using the CRISPR-Cas9 library. Infection of these cells with the "killer" PV system permitted positive selection of cells refractory to viral infection and, through next generation sequencing, identification of a number of factors involved in CHIKV entry. Matrix metalloproteinases were identified as novel entry factors and demonstrated that MMP-targeting drugs efficiently inhibit PV and authentic CHIKV infections. These results suggest this platform holds great promise as a pandemic-preparedness tool that increases the capacity and speed of screening for cell entry factors. It is also a safe platform able to dissect the gene network involved in virus entry. Ceudovitox will help identify new therapeutic targets, thereby aiding the development of treatments against future outbreaks or pandemic pathogens and making a significant contribution to the "100 days" mission. Author SummaryThe increased threat of the emergence of new viral pathogens with pandemic potential highlights the importance of developing scalable high-throughput screening platforms to quickly characterise new emergent viral pathogens. Improving understanding of virus-host interactions, including how viruses enter cells, can help fast development of new targeted therapeutics and reduce pandemic burdens. Current approaches rely on using live viruses, which are highly infectious and must be manipulated in high containment facilities, which are slow and cumbersome. Our new screening platform, Ceudovitox, is a safer, faster and more scalable alternative. Through using pseudotyped viruses, which can only undergo one replication cycle, we are able to distinguish virus entry factors of high containment viruses within highly abundant low containment facilities, allowing us to quickly characterise potential entry factors of high containment viruses. Within this study, Ceudovitox was shown to recognise both known and novel entry factors of Chikungunya virus; a virus which causes chronic arthralgia and arthritis. Matrix metalloproteinases were amongst novel entry factors identified and were seen to reduce virus infection in both pseudotyped and authentic virus assays, demonstrating how Ceudovitox can correctly discover virus entry factors of live high containment viruses. Ceudovitoxs utility as a screening platform for high containment viruses and new emerging pathogens, has the potential increase understanding and speed up the development of new therapeutics during pandemics, helping to reach the 100 Day Mission of pandemic preparedness.

molecular biology↗

bioRxiv2025

Desiccation-induced DNA damage facilitates drug resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen that depends on its ability to spread from host-to-host to survive as a species. Yet, knowledge of transmission-specific traits remains lacking. Here, we report the discovery of a specific adaptive response to desiccation, a stress intrinsically linked to the generation of the aerosol droplets within which Mtb transmits. We show that desiccation inflicts oxidative damage and activates Mtbs DNA repair responses but that this repair is imperfect and results in mutations. We further show that activation of these DNA repair responses is accompanied by increased expression of the transcription-coupled repair factor, mfd, but that this expression serves to buffer the fitness cost of specific resistance-conferring mutations in rpoB, the target of the frontline drug rifampin, rather than to facilitate transcription-coupled DNA repair. Silencing mfd during aerosolization impairs survival of strains harboring the rifampin resistance allele S450L. This function is further supported by whole genome sequence data from over 50,000 clinically circulating strains. These studies indicate that Mtb has evolved transmission-specific stress responses that have enabled it to leverage desiccation-induced DNA damage as a potential source of genetic diversification and drug resistance.

microbiology↗

bioRxiv2024

RapA opens the RNA polymerase clamp to disrupt post-termination complexes and prevent cytotoxic R-loop formation

Following transcript release during intrinsic termination, Escherichia coli RNA polymerase (RNAP) often remains associated with DNA in a post-termination complex (PTC). RNAPs in PTCs are removed from the DNA by the Swi2/Snf2 ATPase RapA. Here, we determined PTC structures on negatively-supercoiled DNA as well as of RapA engaged to dislodge the PTC. We found that core RNAP in the PTC can unwind DNA and initiate RNA synthesis but is prone to producing R-loops. We show that RapA helps control cytotoxic R-loop formation in vivo, likely by disrupting PTCs. Nucleotide binding to RapA triggers a conformational change that opens the RNAP clamp, allowing DNA in the RNAP cleft to reanneal and dissociate. We suggest that analagous ATPases acting on PTCs to suppress transcriptional noise and R-loop formation may be widespread. These results hold significance for the bacterial transcription cycle and highlight a role for RapA in maintaining genome stability.

biophysics↗

bioRxiv2024

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

microbiology↗

bioRxiv2023

Structural and functional insights into the enzymatic plasticity of the SARS-CoV-2 NiRAN Domain

The enzymatic activity of the SARS-CoV-2 nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N-terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5-cap formation, while the role of NMPylation is unclear. The structural mechanisms for these diverse enzymatic activities have not been properly delineated. Here we determine high-resolution cryo-electron microscopy structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions, revealing diverse nucleotide binding poses and divalent metal ion coordination sites to promote its repertoire of activities. The deRNAylation/capping structure explains why GDP is a preferred substrate for the capping reaction over GTP. Altogether, these findings enhance our understanding of the promiscuous coronaviral NiRAN domain, a therapeutic target, and provide an accurate structural platform for drug development.

biophysics↗

bioRxiv2023

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis, an infectious disease that inflicts major health and economic costs around the world1. Mtb encounters a diversity of environments during its lifecycle, and responds to these changing environments by reprogramming its transcriptional output2. However, the transcriptomic features of Mtb remain poorly characterized. In this work, we comprehensively profile the Mtb transcriptome using the SEnd-seq method that simultaneously captures the 5 and 3 ends of RNA3. Surprisingly, we find that the RNA coverage for most of the Mtb transcription units display a gradual drop-off within a 200-500 nucleotide window downstream of the transcription start site, yielding a massive number of incomplete transcripts with heterogeneous 3 ends. We further show that the accumulation of these short RNAs is mainly due to the intrinsically low processivity of the Mtb transcription machinery rather than trans-acting factors such as Rho. Finally, we demonstrate that transcription-translation coupling plays a critical role in generating full-length protein-coding transcripts in Mtb. In sum, our results depict a mycobacterial transcriptome that is dominated by incomplete RNA products, suggesting a distinctive set of transcriptional regulatory mechanisms that could be exploited for new therapeutics.

microbiology↗

bioRxiv2022

Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger elemental pausing by stabilizing a half-translocated (RNA-not-DNA) state and by promoting RNAP swiveling that other factors can enhance. The universal transcription factor NusG (Spt5 in eukaryotes and archaea) N-terminal domain (NGN) modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs (e.g., Bacillus subtilis) enhance some pauses whereas anti-pausing NusGs (e.g., Escherichia coli) suppress some pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). Using biochemistry and cryo-electron microscopy, we show that MtbNusG is a pro-pausing NusG that captures paused, swiveled RNAP by contacts to the RNAP protrusion and to a nontemplate strand-DNA wedge inserted between the NGN and the RNAP gate loop. On the other hand, we find that anti-pausing E. coli NGN contacts the RNAP gate loop to inhibit swiveling and pausing of MtbRNAP. Using CRISPR-mediated mycobacterial genetics, we show that a pro-pausing NGN is required to support robust mycobacterial growth. Our results define an essential function of NusG in mycobacteria and the structural basis of pro-vs. anti-pausing NusG activity with broad implications for NusG function in all domains of life.

molecular biology↗

bioRxiv2022

Structural basis for substrate selection by the SARS-CoV-2 replicase

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP3,4. Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5 RNA cap6.

biophysics↗

bioRxiv2022

Basis of narrow-spectrum activity of fidaxomicin on gut pathogen Clostridioides difficile

Fidaxomicin (Fdx) is widely used to treat Clostridioides difficile (Cdiff) infections (CDIs), but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains enigmatic. CDIs are a leading cause of nosocomial deaths. Fdx, which inhibits RNA polymerase (RNAP), targets Cdiff with minimal effects on gut commensals, reducing CDI recurrence. Here, we present the cryo-electron microscopy structure of Cdiff RNAP in complex with Fdx, allowing us to identify a crucial Fdx-binding determinant of Cdiff RNAP that is absent in most gut microbiota like Proteobacteria and Bacteroidetes. By combining structural, biochemical, and bioinformatic analyses, we establish that a single RNAP residue is a sensitizing element for Fdx narrow-spectrum activity. Our results provide a blueprint for targeted drug design against an important human pathogen.

microbiology↗

bioRxiv2021

Birnaviridae virus factories show features of liquid-liquid phase separation, and are distinct from paracrystalline arrays of virions observed by electron microscopy

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs co-localized with 5-ethynyl uridine in the presence of actinomycin D, confirming they were the site of de novo RNA synthesis, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 seconds, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower co-localization of the VF GFP signal with the capsid protein VP2 (Manders coefficient (MC) 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs are sites of de novo RNA synthesis, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. ImportanceMembers of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

microbiology↗

bioRxiv2021

Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex, or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backwards on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here, we use cryo-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results suggest a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

biophysics↗

bioRxiv2021

An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15

Using available cryo-EM and x-ray crystal structures of the nonstructural proteins that are responsible for SARS-CoV-2 viral RNA replication and transcription, we have constructed an atomistic model of how the proteins assemble into a functioning superstructure. Our principal finding is that the complex is hexameric, centered around nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, where nsp14 is seen to undergo a large conformational change between its two domains. This conformational change facilitates binding of six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Two of the six polymerase subunits are each proposed to carry dimers of nsp13, while two others are proposed to carry monomers. The polymerase subunits that coordinate nsp13 dimers also bind the nucleocapsid, which positions the 5-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analyzing the path of the viral RNA indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping (the nsp12 NiRAN and the nsp14 and nsp16 methyltransferases). The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping and the role of the endonuclease. It provides a platform to guide biochemical and structural research to address the stoichiometric and spatial configuration of the replication-transcription complex. Author SummaryThe replication of the coronavirus genome and the synthesis of subgenomic mRNA is a complex process involving multiple viral proteins. Despite a fairly complete structural picture of the individual proteins that are believed to coalesce into a larger replication-transcription complex, there is no clear model of how these proteins interact. Here we present the first detailed atomistic model of a complete replication-transcription complex for SARS-CoV-2, made up of the non-structural proteins nsp7-nsp16, as well as the nucleocapsid. Forming a large, hexameric superstructure centered around nsp15, the model provides new perspective on the function of its individual components, including the exonuclease, the endonuclease, the NiRAN site, the helicase, the multiple zinc fingers, and the nucleocapsid. It offers a cohesive view of replication, proofreading, template switching and mRNA capping, which should serve as a guide for future experimental exploration.

molecular biology↗

bioRxiv2021

Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals. Significance StatementThe COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.

biochemistry↗

bioRxiv2020

Inhibition of AlgU by MucA is required for viability in Pseudomonas aeruginosa

During decades-long infections in the cystic fibrosis (CF) airway, Pseudomonas aeruginosa undergoes selection. One bacterial genetic adaptation often observed in CF isolates is mucA mutations. MucA inhibits the sigma factor AlgU. Mutations in mucA lead to AlgU misregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. Due to its ability to be mutated, mucA is assumed to be dispensable for bacterial viability. Here we show that, paradoxically, a portion of mucA is essential in P. aeruginosa. We demonstrate that mucA is no longer required in a strain lacking algU, that mucA alleles encoding for proteins that do not bind to AlgU are insufficient for viability, and that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Furthermore, we found that overexpression of algU prevents cell growth in the absence of MucA, and that this phenotype can be rescued by overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity results in the loss of bacterial viability. Finally, we speculate that essentiality of anti-sigma factors that regulate envelope function may be a widespread phenomenon in bacteria.

microbiology↗

bioRxiv2020

Structural basis for transcription complex disruption by the Mfd translocase

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (EC). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.

biophysics↗