RESUMO
Human mitochondria harbour a circular, polyploid genome (mtDNA) encoding 11 messenger RNAs (mRNAs), two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). Mitochondrial transcription produces long, polycistronic transcripts that span almost the entire length of the genome, and hence contain all three types of RNAs. The primary transcripts then undergo a number of processing and maturation steps, which constitute key regulatory points of mitochondrial gene expression. The first step of mitochondrial RNA processing consists of the separation of primary transcripts into individual, functional RNA molecules and can occur by two distinct pathways. Both are carried out by dedicated molecular machineries that substantially differ from RNA processing enzymes found elsewhere. As a result, the underlying molecular mechanisms remain poorly understood. Over the last years, genetic, biochemical and structural studies have identified key players involved in both RNA processing pathways and provided the first insights into the underlying mechanisms. Here, we review our current understanding of RNA processing in mammalian mitochondria and provide an outlook on open questions in the field.
Assuntos
DNA Mitocondrial , Mitocôndrias , Processamento Pós-Transcricional do RNA , RNA Mitocondrial , Humanos , DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Transcrição Gênica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.
Assuntos
RNA Polimerases Dirigidas por DNA , RNA de Cloroplastos , RNA de Cloroplastos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Regulação da Expressão Gênica de Plantas , Transcrição GênicaRESUMO
Single-particle cryoelectron microscopy (cryo-EM) allows structure determination of large macromolecular complexes from conformationally and compositionally heterogeneous mixtures of particles. This technique has been used to reveal the architecture of the mitochondrial ribosome and to visualize transient states that occur during the translation cycle or during mitoribosome biogenesis. Here, we outline an exemplary workflow for the analysis of single-particle cryo-EM data of human mitoribosome samples. In addition, we provide an example dataset which can be used for training purposes alongside the protocol.
Assuntos
Ribossomos Mitocondriais , Imagem Individual de Molécula , Humanos , Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Processamento de Imagem Assistida por Computador/métodosRESUMO
Multifunctional proteins are challenging as it can be difficult to confirm pathomechanisms associated with disease-causing genetic variants. The human 17ß-hydroxysteroid dehydrogenase 10 (HSD10) is a moonlighting enzyme with at least two structurally and catalytically unrelated functions. HSD10 disease was originally described as a disorder of isoleucine metabolism, but the clinical manifestations were subsequently shown to be linked to impaired mtDNA transcript processing due to deficient function of HSD10 in the mtRNase P complex. A surprisingly large number of other, mostly enzymatic and potentially clinically relevant functions have been attributed to HSD10. Recently, HSD10 was reported to exhibit phospholipase C-like activity towards cardiolipins (CL), important mitochondrial phospholipids. To assess the physiological role of the proposed CL-cleaving function, we studied CL architectures in living cells and patient fibroblasts in different genetic backgrounds and lipid environments using our well-established LC-MS/MS cardiolipidomic pipeline. These experiments revealed no measurable effect on CLs, indicating that HSD10 does not have a physiologically relevant function towards CL metabolism. Evolutionary constraints could explain the broad range of reported substrates for HSD10 in vitro. The combination of an essential structural with a non-essential enzymatic function in the same protein could direct the evolutionary trajectory towards improvement of the former, thereby increasing the flexibility of the binding pocket, which is consistent with the results presented here.
Assuntos
3-Hidroxiacil-CoA Desidrogenases , Hidroxiesteroide Desidrogenases , Humanos , 3-Hidroxiacil-CoA Desidrogenases/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Cardiolipinas , Cromatografia Líquida , Espectrometria de Massas em Tandem , DNA Mitocondrial , Fosfolipases Tipo CRESUMO
Aminoacylation of transfer RNA (tRNA) is a key step in protein biosynthesis, carried out by highly specific aminoacyl-tRNA synthetases (ARSs). ARSs have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl-tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl-tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N-terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1-related syndrome and define an emerging disease spectrum: ARS-related developmental disorders with or without microcephaly.
Assuntos
Aminoacil-tRNA Sintetases , Doença de Charcot-Marie-Tooth , Microcefalia , Triptofano-tRNA Ligase , Animais , Humanos , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , Ligases , Microcefalia/genética , Microcefalia/patologia , RNA de Transferência , Triptofano-tRNA Ligase/genética , Peixe-Zebra/genéticaRESUMO
Ribonuclease P (RNase P) enzymes are responsible for the 5' processing of tRNA precursors. In addition to the well-characterised ribozyme-based RNase P enzymes, an evolutionarily distinct group of protein-only RNase Ps exists. These proteinaceous RNase Ps (PRORPs) can be found in all three domains of life and can be divided into two structurally different types: eukaryotic and prokaryotic. Recent structural studies on members of both families reveal a surprising diversity of molecular architectures, but also highlight conceptual and mechanistic similarities. Here, we provide a comparison between the different types of PRORP enzymes and review how the combination of structural, biochemical, and biophysical studies has led to a molecular picture of protein-mediated tRNA processing.
Assuntos
Arabidopsis , RNA Catalítico , Arabidopsis/genética , Humanos , Processamento Pós-Transcricional do RNA , RNA Catalítico/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/química , Ribonuclease P/genética , Ribonuclease P/metabolismoRESUMO
Final maturation steps during ribosome biogenesis require the assistance of assembly and quality control factors to ensure the folding of rRNA and proteins into a functional translation machinery. Here we integrate several recent structural snapshots of native large ribosomal subunit intermediates into the complex pathway of mitochondrial ribosome assembly.
Assuntos
Ribossomos Mitocondriais , Ribossomos , Domínio Catalítico , Humanos , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Biogênese de Organelas , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismoRESUMO
Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Anticódon/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , RNA Fúngico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease P/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.
Assuntos
SARS-CoV-2/enzimologia , Dimerização , Humanos , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.
Assuntos
COVID-19/prevenção & controle , Citidina/análogos & derivados , Hidroxilaminas/metabolismo , Mutagênese/genética , RNA Viral/genética , SARS-CoV-2/genética , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Sequência de Bases , COVID-19/virologia , Citidina/química , Citidina/metabolismo , Citidina/farmacologia , Humanos , Hidroxilaminas/química , Hidroxilaminas/farmacologia , Modelos Moleculares , Estrutura Molecular , Mutagênese/efeitos dos fármacos , Mutação/efeitos dos fármacos , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Tratamento Farmacológico da COVID-19RESUMO
Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.
Assuntos
Proteínas de Ligação ao GTP/química , Ribossomos Mitocondriais/química , Proteínas Monoméricas de Ligação ao GTP/química , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Biogênese de Organelas , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Dobramento de Proteína , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores/química , Subunidades Ribossômicas Maiores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Coronaviruses use an RNA-dependent RNA polymerase (RdRp) to replicate and express their genome. The RdRp associates with additional non-structural proteins (nsps) to form a replication-transcription complex (RTC) that carries out RNA synthesis, capping and proofreading. However, the structure of the RdRp long remained elusive, thus limiting our understanding of coronavirus genome expression and replication. Recently, the cryo-electron microscopy structure of SARS-CoV-1 RdRp was reported. Driven by the ongoing COVID-19 pandemic, structural data on the SARS-CoV-2 polymerase and associated factors has since emerged at an unprecedented pace, with more than twenty structures released to date. This review provides an overview of the currently available coronavirus RdRp structures and outlines how they have, together with functional studies, led to a molecular understanding of the viral polymerase, its interactions with accessory factors and the mechanisms by which promising antivirals may inhibit coronavirus replication.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5' capping and 3' polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3'-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3'-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.
Assuntos
Proteínas de Membrana/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões 3' não Traduzidas , Genes Mitocondriais , Proteínas de Membrana/química , Proteínas de Membrana/genética , Motivos de Nucleotídeos , Ligação Proteica , Domínios Proteicos , RNA Mensageiro/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Coronaviruses use an RNA-dependent RNA polymerase to replicate and transcribe their RNA genome. The structure of the SARS-CoV-2 polymerase was determined by cryo-electron microscopy within a short time in spring 2020. The structure explains how the viral enzyme synthesizes RNA and how it replicates the exceptionally large genome in a processive manner. The most recent structure-function studies further reveal the mechanism of polymerase inhibition by remdesivir, an approved drug for the treatment of COVID-19.
RESUMO
Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , RNA Polimerase Dependente de RNA/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , Aptâmeros de Nucleotídeos , RNA-Polimerase RNA-Dependente de Coronavírus/efeitos dos fármacos , Nucleotídeos , RNA Viral , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2/enzimologia , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Microscopia Crioeletrônica , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Mitocondriais/efeitos dos fármacos , Humanos , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Especificidade por Substrato/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes1-3. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles'. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses3. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19)4.
Assuntos
Betacoronavirus/enzimologia , Microscopia Crioeletrônica , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Betacoronavirus/ultraestrutura , RNA-Polimerase RNA-Dependente de Coronavírus , Modelos Moleculares , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/ultraestrutura , SARS-CoV-2 , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/ultraestruturaRESUMO
Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m7G-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Metiltransferases/química , Complexos Multienzimáticos/química , Nucleotidiltransferases/química , Monoéster Fosfórico Hidrolases/química , Vaccinia virus/ultraestrutura , Proteínas Virais/química , Microscopia Crioeletrônica , Complexos Multienzimáticos/ultraestrutura , RNA Mensageiro/química , Imagem Individual de Molécula , Transcrição Gênica , Vaccinia virus/genética , Vaccinia virus/metabolismoRESUMO
Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.