Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.

Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to cause massive global upheaval. Coronaviruses are positive-strand RNA viruses with an unusually large genome of ~30 kb. They express an RNA-dependent RNA polymerase and a cohort of other replication enzymes and supporting factors to transcribe and replicate their genomes. The proteins performing these essential processes are prime antiviral drug targets, but drug discovery is hindered by our incomplete understanding of coronavirus RNA synthesis and processing. In infected cells, the RNA-dependent RNA polymerase must coordinate with other viral and host factors to produce both viral mRNAs and new genomes. Recent research aiming to decipher and contextualize the structures, functions and interplay of the subunits of the SARS-CoV-2 replication and transcription complex proteins has burgeoned. In this Review, we discuss recent advancements in our understanding of the molecular basis and complexity of the coronavirus RNA-synthesizing machinery. Specifically, we outline the mechanisms and regulation of RNA translation, replication and transcription. We also discuss the composition of the replication and transcription complexes and their suitability as targets for antiviral therapy.

AlphaFold: Research accelerator and hypothesis generator.

To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses.

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 cryoelectron microscopy (cryo-EM) 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.

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 pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome.

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide1. Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output2. However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells3 to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5' ends aligned at transcription start sites and 3' ends located 200-500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription-translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics.

Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

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 analogues, 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 analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-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 adenosine triphosphate3,4. Our results explain 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 (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Structural basis of transcriptional activation by the Mycobacterium tuberculosis intrinsic antibiotic-resistance transcription factor WhiB7.

In pathogenic mycobacteria, transcriptional responses to antibiotics result in induced antibiotic resistance. WhiB7 belongs to the Actinobacteria-specific family of Fe-S-containing transcription factors and plays a crucial role in inducible antibiotic resistance in mycobacteria. Here, we present cryoelectron microscopy structures of Mycobacterium tuberculosis transcriptional regulatory complexes comprising RNA polymerase σA-holoenzyme, global regulators CarD and RbpA, and WhiB7, bound to a WhiB7-regulated promoter. The structures reveal how WhiB7 interacts with σA-holoenzyme while simultaneously interacting with an AT-rich sequence element via its AT-hook. Evidently, AT-hooks, rare elements in bacteria yet prevalent in eukaryotes, bind to target AT-rich DNA sequences similarly to the nuclear chromosome binding proteins. Unexpectedly, a subset of particles contained a WhiB7-stabilized closed promoter complex, revealing this intermediate's structure, and we apply kinetic modeling and biochemical assays to rationalize how WhiB7 activates transcription. Altogether, our work presents a comprehensive view of how WhiB7 serves to activate gene expression leading to antibiotic resistance.

Basis of narrow-spectrum activity of fidaxomicin on Clostridioides difficile.

Fidaxomicin (Fdx) is widely used to treat Clostridioides difficile (Cdiff) infections, but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains unknown. Cdiff infections are a leading cause of nosocomial deaths1. Fidaxomicin, which inhibits RNA polymerase, targets Cdiff with minimal effects on gut commensals, reducing recurrence of Cdiff infection2,3. Here we present the cryo-electron microscopy structure of Cdiff RNA polymerase in complex with fidaxomicin and identify a crucial fidaxomicin-binding determinant of Cdiff RNA polymerase that is absent in most gut microbiota such as Proteobacteria and Bacteroidetes. By combining structural, biochemical, genetic and bioinformatic analyses, we establish that a single residue in Cdiff RNA polymerase is a sensitizing element for fidaxomicin narrow-spectrum activity. Our results provide a blueprint for targeted drug design against an important human pathogen.

Stepwise Promoter Melting by Bacterial RNA Polymerase.

Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.

A general mechanism for transcription bubble nucleation in bacteria.

Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ70, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ70. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σN-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σN, like σ70, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.

Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC (Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

Structural basis of dual activation of cell division by the actinobacterial transcription factors WhiA and WhiB.

Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as Mycobacterium tuberculosis. The WhiA/B regulons and binding sites have been elucidated in Streptomyces venezuelae (Sven), where they coordinate to activate sporulation septation. However, how these factors cooperate at the molecular level is not understood. Here we present cryoelectron microscopy structures of Sven transcriptional regulatory complexes comprising RNA polymerase (RNAP) σA-holoenzyme and WhiA and WhiB, in complex with the WhiA/B target promoter sepX. These structures reveal that WhiB binds to domain 4 of σA (σA4) of the σA-holoenzyme, bridging an interaction with WhiA while making non-specific contacts with the DNA upstream of the -35 core promoter element. The N-terminal homing endonuclease-like domain of WhiA interacts with WhiB, while the WhiA C-terminal domain (WhiA-CTD) makes base-specific contacts with the conserved WhiA GACAC motif. Notably, the structure of the WhiA-CTD and its interactions with the WhiA motif are strikingly similar to those observed between σA4 housekeeping σ-factors and the -35 promoter element, suggesting an evolutionary relationship. Structure-guided mutagenesis designed to disrupt these protein-DNA interactions reduces or abolishes developmental cell division in Sven, confirming their significance. Finally, we compare the architecture of the WhiA/B σA-holoenzyme promoter complex with the unrelated but model CAP Class I and Class II complexes, showing that WhiA/WhiB represent a new mechanism in bacterial transcriptional activation.

Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein.

Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively "mark" antiparallel overlap in dynamic cytoskeletal networks.

Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding.

A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex1-3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed4-6. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.

6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase.

Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ70-holoenzyme (Eσ70) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσ70 determined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3' endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσ70 and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP.

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 severe acute respiratory syndrome coronavirus 2 (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 cross-linking, 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 nucleoside triphosphate (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.

Rewiring the specificity of extracytoplasmic function sigma factors.

Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid-nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

The antibiotic sorangicin A inhibits promoter DNA unwinding in a Mycobacterium tuberculosis rifampicin-resistant RNA polymerase.

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (RifR) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of RifR RNAPs, including the most prevalent clinical RifR RNAP substitution found in Mtb infected patients (S456>L of the ß subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.