RNA Polymerase Complex Research Articles

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli.

To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome's transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451).

Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus

PB1 functions as the catalytic subunit of influenza virus RNA polymerase complex and plays an essential role in viral RNA transcription and replication. To determine plasticity in the PB1 enzymatic site and map catalytically important residues, 658 mutants were constructed, each with one to seven mutations in the enzymatic site of PB1. The polymerase activities of these mutants were quantified using a minigenome assay, and polymerase activity-associated residues were identified using sparse learning. Results showed that polymerase activities are affected by the residues not only within the conserved motifs, but also across the inter-motif regions of PB1, and the latter are primarily located at the base of the palm domain, a region that is conserved in avian PB1 but with high sequence diversity in swine PB1. Our results suggest that mutations outside the PB1 conserved motifs may affect RNA replication and could be associated with influenza virus host adaptation.

Therapeutic targeting of measles virus polymerase with ERDRP-0519 suppresses all RNA synthesis activity.

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.

In Silico Investigations on Switching Promotor Recognition of Phage RNA Polymerase Variants Following Path Along Lab Directed Evolution

In this work, we computationally studied how a viral RNA polymerase (RNAP) from bacteriophage T7 under lab-directed evolution pressure evolves into mutant variants that switch the RNAP function on recognition of T7 promoter into T3 promoter. We first constructed a closed initiation complex of T7 RNAP via docking an apo RNAP onto ∼30 bp DNA, and then built up all-atom simulation systems for both the wild-type (wt) T7 RNAP and a series of mutant (mt) RNAPs, with each of them in complex with the T7 vs T3 promoter for comparison.

Hepatitis C Virus Protease Inhibitors Suppress Virus Replication and Act Synergistically with the SARS-CoV2 Polymerase Inhibitor Remdesivir

The COVID-19 pandemic has caused > 1 million deaths worldwide, and crippled the global economy. The current antiviral standard of care for COVID-19 is remdesivir, which inhibits the viral polymerase. The two viral proteases, the Main protease (Mpro) and the papain-like protease (PLpro), are also attractive as antiviral targets, particularly because cleavage of the two viral polyproteins by these two proteases generates proteins required for the assembly and/or activity of the viral RNA polymerase complex.

Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication

Remdesivir is a nucleoside analog approved by the US FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryo-EM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of 3 copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of 3 bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

Structural Basis of SARS-CoV-2 Polymerase Inhibition by Favipiravir

SummaryThe outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into an unprecedented global pandemic. Nucleoside analogs, such as Remdesivir and Favipiravir, can serve as the first-line broad-spectrum antiviral drugs by targeting the viral polymerases. However, the underlying mechanisms for the antiviral efficacies of these drugs are far from well understood. Here, we reveal that Favipiravir, as a pyrazine derivative, could be incorporated into the viral RNA products by mimicking both adenine and guanine nucleotides. This drug thus inhibits viral replication mainly by inducing mutations in progeny RNAs, different from Remdesivir or other RNA-terminating nucleoside analogs that impair the elongation of RNA products. We further determined the cryo-EM structure of Favipiravir bound to the replicating polymerase complex of SARS-CoV-2 in the pre-catalytic state. This structure provides a missing snapshot for visualizing the catalysis dynamics of coronavirus polymerase, and reveals an unexpected base-pairing pattern between Favipiravir and pyrimidine residues that may explain its capacity for mimicking both adenine and guanine nucleotides. These findings shed light on the mechanism of coronavirus polymerase catalysis and provide a rational basis for developing antiviral drugs to combat the SARS-CoV-2 pandemic.

A tight couple makes messenger RNAs

RNA Splicing Gene expression in eukaryotes first requires transcription of DNA to an RNA copy and then splicing to form the final, processed messenger RNA (mRNA). Zhang et al. investigated how gene transcription and RNA splicing are physically coupled. Using cryo–electron microscopy, they resolved the molecular structure of a complex of the transcription enzyme RNA polymerase II with part of the splicing machinery, the U1 small nuclear ribonucleoprotein particle. The results provide important details for our understanding of coupled mRNA production. Science , this issue p. [305][1] [1]: /lookup/doi/10.1126/science.abf1870

Model of the RNA Polymerase Complex of the SARS-CoV-2 Virus with Favipiravir

A model of a multidomain complex is constructed using molecular modeling methods to explain the mechanism of the inhibitory effect of favipiravir on RNA-dependent RNA polymerase (RdRp) of the SARS-CoV-2 coronavirus. As the initial atomic coordinates, we use cryoelectron microscopy data for the apo form of RdRp of the SARS-CoV-2 virus and data on the structure of RdRp of the hepatitis C virus. After appropriate substitutions, an RdRp complex containing RNA chains and a potential enzyme inhibitor, favipiravir in the form of ribosatriphosphate, are constructed. The structure of the complex in aqueous shells, which includes more than 100 000 atoms, is optimized by molecular dynamics methods. Analysis of the active site with the incorporated favipiravir molecule makes it possible to explain the chemical reaction of the enzyme with the inhibitor.

ALM1, encoding a Fe-superoxide dismutase, is critical for rice chloroplast biogenesis and drought stress response

Chloroplasts are the center of plant life activities including photosynthesis, growth and development, and abiotic stress response. Chloroplast development and biogenesis in rice have been studied in detail, but how does abiotic stress affect chloroplasts is less studied. We obtained an albino mutant, alm1, whose chlorophyll content was greatly decreased. Transmission electron microscopy showed that chloroplast development in alm1 was blocked, especially in thylakoid-like structures, which could not form normally. The ALM1 gene encodes a chloroplast-localized superoxide dismutase. Full-length ALM1 successfully restored the non-albino phenotype, and in knockout lines, the albino phenotype reappeared. The ALM1 gene is expressed mainly in young leaves. alm1 plants died as a consequence of excessive reactive oxygen accumulation after the third-leaf stage. A series of biochemical assays verified that ALM1 interacted with the OsTrxz protein, which is one of the components of plastid-encoded RNA polymerase (PEP) complexes. A western blot experiment indicated that ALM1 played an important role in stabilizing OsTrxz in rice. An overexpression test of ALM1 revealed that ALM1 can increase drought resistance by removing excess reactive oxygen in rice seedlings. This study suggests that ALM1 not only participates in rice chloroplast biogenesis, but also increases rice stress resistance by scavenging excess reactive oxygen.

Arabidopsis Seedling Lethal 1 Interacting With Plastid-Encoded RNA Polymerase Complex Proteins Is Essential for Chloroplast Development.

Mitochondrial transcription termination factors (mTERFs) are highly conserved proteins in metazoans. Plants have many more mTERF proteins than animals. The functions and the underlying mechanisms of plants’ mTERFs remain largely unknown. In plants, mTERF family proteins are present in both mitochondria and plastids and are involved in gene expression in these organelles through different mechanisms. In this study, we screened Arabidopsis mutants with pigment-defective phenotypes and isolated a T-DNA insertion mutant exhibiting seedling-lethal and albino phenotypes [seedling lethal 1 (sl1)]. The SL1 gene encodes an mTERF protein localized in the chloroplast stroma. The sl1 mutant showed severe defects in chloroplast development, photosystem assembly, and the accumulation of photosynthetic proteins. Furthermore, the transcript levels of some plastid-encoded proteins were significantly reduced in the mutant, suggesting that SL1/mTERF3 may function in the chloroplast gene expression. Indeed, SL1/mTERF3 interacted with PAP12/PTAC7, PAP5/PTAC12, and PAP7/PTAC14 in the subgroup of DNA/RNA metabolism in the plastid-encoded RNA polymerase (PEP) complex. Taken together, the characterization of the plant chloroplast mTERF protein, SL1/mTERF3, that associated with PEP complex proteins provided new insights into RNA transcription in the chloroplast.

Remdesivir is a Delayed Translocation Inhibitor of SARS CoV-2 Replication

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugsFunding: This work was supported in part by Welch Foundation grantsF-1604 (to K.A.J.) and F-1938 (to D.W.T.), National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) R01AI110577 (to K.A.J) and National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) R01114223 (to K.A.J.) and R35GM138348 (to D.W.T.), Army Research Office Grant W911NF-15-1-0120(to D.W.T.), and a Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation Medical Research Award (to D.W.T.). D.W.T is a CPRIT Scholar supported by the Cancer Prevention andResearch Institute of Texas (RR160088) and an Army Young Investigator supported by the Army Research Office (W911NF-19-1-0021).Conflict of Interest: All authors declare no competing interests.

Analysis of a photosynthetic cyanobacterium rich in internal membrane systems via gradient profiling by sequencing (Grad-seq).

Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma–antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA–protein complexes and multisubunit protein complexes in a photosynthetic organism.

Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid–liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid–liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

A fully assembled plastid-encoded RNA polymerase complex detected in etioplasts and proplastids in Arabidopsis.

The plastid-encoded genes of higher plants are transcribed by at least two types of RNA polymerases, the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). In mature photosynthesizing leaves, the vast majority of the genes are transcribed by PEP. However, the regulatory mechanisms controlling plastid transcription during early light response is unclear. Chloroplast development is suggested to be associated with a shift in the usage of the primary RNA polymerase from NEP to PEP as the expression of the plastid-encoded photosynthesis genes is induced upon light exposure. Assembly of the PEP complex has been suggested as a rate-limiting step for full activation of plastid-encoded photosynthesis gene expression. However, two sigma factor mutants, sig2 and sig6, with reduced PEP activity, showed significantly lower expression of the plastid-encoded photosynthesis genes already in the dark and during the first hours of light exposure indicating that PEP activity is required for basal expression of plastid-encoded photosynthesis genes in the dark and during early light response. Furthermore, in etioplasts and proplastids a fully assembled PEP complex was revealed on Blue Native PAGE. Our results indicate that a full assembly of the PEP complex is possible in the dark and that PEP drives basal transcriptional activity of plastid-encoded photosynthesis genes in the dark. Assembly of the complex is most likely not a rate-limiting step for full activation of plastid-encoded photosynthesis gene expression which is rather achieved either by the abundance of the PEP complex or by some posttranslational regulation of the individual PEP components.

Type-IInterferon-Inducible SERTAD3 Inhibits Influenza A Virus Replication by Blocking the Assembly of Viral RNA Polymerase Complex

Influenza A virus (IAV) infection stimulates a type I interferon (IFN-I) response in host cells that exerts antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). However, most ISGs are poorly studied for their roles in the infection of IAV. Herein, we demonstrate that SERTA domain containing 3 (SERTAD3) has a significant inhibitory effect on IAV replication invitro. More importantly, Sertad3-/- mice develop more severe symptoms upon IAV infection. Mechanistically, we find SERTAD3 reduces IAV replication through interacting with viral polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acidic protein (PA) to disrupt the formation of the RNA-dependent RNA polymerase (RdRp) complex. We further identify an 8-amino-acid peptide of SERTAD3 as a minimum interacting motif that can disrupt RdRp complex formation and inhibit IAV replication. Thus, our studies not only identify SERTAD3 as an antiviral ISG, but also provide the mechanism of potential application of SERTAD3-derived peptide in suppressing influenza replication.

Comprehensive assessment of amino acid substitutions in the trimeric RNA polymerase complex of influenza A virus detected in clinical trials of baloxavir marboxil.

BackgroundBaloxavir marboxil (BXM) is an approved drug that selectively targets cap‐dependent endonuclease on PA subunit in the RNA polymerase complex of influenza A and B viruses. Amino acid substitutions at position 38 in the PA subunit were identified as a major pathway for reduced susceptibility to baloxavir acid (BXA), the active form of BXM. Additionally, substitutions found at positions E23, A37, and E199 in the PA subunit impact BXA susceptibility by less than 10‐fold.MethodsWe comprehensively evaluated the impact of novel amino acid substitutions identified in PA, PB1, and PB2 subunits in BXM clinical trials and influenza sequence databases by means of drug susceptibility and replicative capacity.ResultsPA/I38N in A(H1N1)pdm09 and PA/I38R in A(H3N2) were newly identified as treatment‐emergent substitutions in the CAPSTONE‐2 study. The I38N substitution conferred reduced susceptibility by 24‐fold, whereas replicative capacity of the I38N‐substituted virus was impaired compared with the wild‐type. The I38R‐substituted virus was not viable in cell culture. All other mutations assessed in this extensive study did not significantly affect BXA susceptibility (< 2.4‐fold change).ConclusionThese results provide additional information on the impact of amino acid substitutions in the trimeric viral polymerase complex to BXA susceptibility and will further support influenza surveillance.

UvrD helicase\u2013RNA polymerase interactions are governed by UvrD\u2019s carboxy-terminal Tudor domain

All living organisms have to cope with the constant threat of genome damage by UV light and other toxic reagents. To maintain the integrity of their genomes, organisms developed a variety of DNA repair pathways. One of these, the Transcription Coupled DNA-Repair (TCR) pathway, is triggered by stalled RNA Polymerase (RNAP) complexes at DNA damage sites on actively transcribed genes. A recently elucidated bacterial TCR pathway employs the UvrD helicase pulling back stalled RNAP complexes from the damage, stimulating recruitment of the DNA-repair machinery. However, structural and functional aspects of UvrD’s interaction with RNA Polymerase remain elusive. Here we used advanced solution NMR spectroscopy to investigate UvrD’s role within the TCR, identifying that the carboxy-terminal region of the UvrD helicase facilitates RNAP interactions by adopting a Tudor-domain like fold. Subsequently, we functionally analyzed this domain, identifying it as a crucial component for the UvrD–RNAP interaction besides having nucleic-acid affinity.

Prediction of rifampicin resistance beyond the RRDR using structure-based machine learning approaches

Rifampicin resistance is a major therapeutic challenge, particularly in tuberculosis, leprosy, P. aeruginosa and S. aureus infections, where it develops via missense mutations in gene rpoB. Previously we have highlighted that these mutations reduce protein affinities within the RNA polymerase complex, subsequently reducing nucleic acid affinity. Here, we have used these insights to develop a computational rifampicin resistance predictor capable of identifying resistant mutations even outside the well-defined rifampicin resistance determining region (RRDR), using clinical M. tuberculosis sequencing information. Our tool successfully identified up to 90.9% of M. tuberculosis rpoB variants correctly, with sensitivity of 92.2%, specificity of 83.6% and MCC of 0.69, outperforming the current gold-standard GeneXpert-MTB/RIF. We show our model can be translated to other clinically relevant organisms: M. leprae, P. aeruginosa and S. aureus, despite weak sequence identity. Our method was implemented as an interactive tool, SUSPECT-RIF (StrUctural Susceptibility PrEdiCTion for RIFampicin), freely available at https://biosig.unimelb.edu.au/suspect_rif/.

Mutational analysis of Escherichia coli GreA protein reveals new functional activity independent of antipause and lethal when overexpressed

There is a growing appreciation for the diverse regulatory consequences of the family of proteins that bind to the secondary channel of E. coli RNA polymerase (RNAP), such as GreA, GreB or DksA. Similar binding sites could suggest a competition between them. GreA is characterised to rescue stalled RNAP complexes due to its antipause activity, but also it is involved in transcription fidelity and proofreading. Here, overexpression of GreA is noted to be lethal independent of its antipause activity. A library of random GreA variants has been used to isolate lethality suppressors to assess important residues for GreA functionality and its interaction with the RNA polymerase. Some mutant defects are inferred to be associated with altered binding competition with DksA, while other variants seem to have antipause activity defects that cannot reverse a GreA-sensitive pause site in a fliC::lacZ reporter system. Surprisingly, apparent binding and cleavage defects are found scattered throughout both the coiled-coil and globular domains. Thus, the coiled-coil of GreA is not just a measuring stick ensuring placement of acidic residues precisely at the catalytic centre but also seems to have binding functions. These lethality suppressor mutants may provide valuable tools for future structural and functional studies.