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1.
Science ; 369(6503): 554-557, 2020 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-32732422

RESUMEN

Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae We combined whole-cell cross-linking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.


Asunto(s)
Mycoplasma pneumoniae/metabolismo , Mycoplasma pneumoniae/ultraestructura , Extensión de la Cadena Peptídica de Translación , Mapas de Interacción de Proteínas , Transcripción Genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Genoma Bacteriano , Humanos , Mycoplasma pneumoniae/genética , Factores de Elongación de Péptidos/metabolismo , Ribosomas/metabolismo , Transcriptoma
3.
Phys Rev Lett ; 125(4): 048104, 2020 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-32794805

RESUMEN

The RNA world scenario posits replication by RNA polymerases. On early Earth, a geophysical setting is required to separate hybridized strands after their replication and to localize them against diffusion. We present a pointed heat source that drives exponential, RNA-catalyzed amplification of short RNA with high efficiency in a confined chamber. While shorter strands were periodically melted by laminar convection, the temperature gradient caused aggregated polymerase molecules to accumulate, protecting them from degradation in hot regions of the chamber. These findings demonstrate a size-selective pathway for autonomous RNA-based replication in natural nonequilibrium conditions.


Asunto(s)
Ecosistema , ARN/química , ARN/genética , Catálisis , ADN/química , ADN/genética , ADN/metabolismo , Replicación del ADN , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Planeta Tierra , Evolución Molecular , Calor , Biosíntesis de Proteínas/genética , ARN/metabolismo
4.
Nat Commun ; 11(1): 3392, 2020 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-32636376

RESUMEN

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , G-Cuádruplex , Estructuras R-Loop , Transcripción Genética , Proteínas Virales/genética , ADN/análisis , ADN/química , ARN Polimerasas Dirigidas por ADN/química , Transferencia Resonante de Energía de Fluorescencia , Unión Proteica , ARN/química , ARN Mensajero/química , Sitio de Iniciación de la Transcripción , Proteínas Virales/química
5.
PLoS Pathog ; 16(7): e1008672, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32706832

RESUMEN

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level ß-lactam resistance (oxacillin MIC 2-4 µg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 µg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit ß) or rpoC (RNA polymerase subunit ß') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.


Asunto(s)
Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Regulación Bacteriana de la Expresión Génica/genética , Staphylococcus aureus Resistente a Meticilina/genética , Proteínas de Unión a las Penicilinas/genética , Resistencia betalactámica/genética , Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos
8.
Proc Natl Acad Sci U S A ; 117(27): 15642-15649, 2020 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-32571927

RESUMEN

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , ADN/química , ARN/química , Transcripción Genética , Dominio Catalítico , ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/ultraestructura , Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Nucleótidos , Conformación Proteica , Pliegue de Proteína , ARN/genética , Imagen Individual de Molécula
9.
Nat Commun ; 11(1): 2728, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-32483114

RESUMEN

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.


Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Conformación Proteica , Multimerización de Proteína , Transactivadores/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Fenol/metabolismo , Unión Proteica , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Homología de Secuencia de Aminoácido , Transactivadores/genética , Transactivadores/metabolismo
10.
Biochemistry (Mosc) ; 85(5): 523-530, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32571182

RESUMEN

In the pathogenesis of the infectious process in the respiratory tract by SARS, MERS, and COVID-19 coronaviruses, two stages can be distinguished: early (etiotropic) and late (pathogenetic) ones. In the first stage, when the virus multiplication and accumulation are prevalent under insufficient host immune response, the use of chemotherapeutic agents blocking the reproduction of the virus is reasonable to suppress the development of the disease. This article considers six major chemotherapeutic classes aimed at certain viral targets: inhibitors of viral RNA polymerase, inhibitors of viral protease Mpro, inhibitors of proteolytic activation of viral protein S allowing virus entry into the target cell, inhibitors of virus uncoating in cellular endosomes, compounds of exogenous interferons, and compounds of natural and recombinant virus-neutralizing antibodies. In the second stage, when the multiplication of the virus decreases and threatening pathological processes of excessive inflammation, acute respiratory distress syndrome, pulmonary edema, hypoxia, and secondary bacterial pneumonia and sepsis events develop, a pathogenetic therapeutic approach including extracorporeal blood oxygenation, detoxification, and anti-inflammatory and anti-bacterial therapy seems to be the most effective way for the patient's recovery.


Asunto(s)
Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Neumonía Viral/tratamiento farmacológico , Anticuerpos Antivirales/uso terapéutico , Antivirales/farmacología , Betacoronavirus/enzimología , Betacoronavirus/inmunología , Infecciones por Coronavirus/virología , Cisteína Endopeptidasas , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Quimioterapia Combinada , Humanos , Interferón alfa-2/uso terapéutico , Pandemias , Neumonía Viral/virología , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos
11.
PLoS Negl Trop Dis ; 14(6): e0008283, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32497085

RESUMEN

BACKGROUND: The Crimean-Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity. METHODOLOGY/PRINCIPLE FINDINGS: Recombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2'-deoxy-2'-fluoro-CTP (FdC) and 2'-deoxy-2'-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, the ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions. CONCLUSIONS/SIGNIFICANCE: The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , Enzimas Desubicuitinizantes/fisiología , Virus de la Fiebre Hemorrágica de Crimea-Congo/enzimología , Replicación Viral/efectos de los fármacos , Amidas/farmacología , Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Fiebre Hemorrágica de Crimea/virología , Humanos , Mutación , Estructura Terciaria de Proteína , Pirazinas/farmacología , ARN Viral , Ribavirina/farmacología
12.
Nat Commun ; 11(1): 3122, 2020 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-32561742

RESUMEN

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Espectrometría de Masas , Mutación , Mapeo de Interacción de Proteínas , Precursores del ARN/metabolismo , Estabilidad del ARN , RNA-Seq , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por Sustrato/genética
13.
Cells ; 9(5)2020 05 20.
Artículo en Inglés | MEDLINE | ID: covidwho-324261

RESUMEN

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Neumonía Viral/virología , ARN Viral/metabolismo , Replicación Viral/fisiología , Animales , Dominio Catalítico , ARN Polimerasas Dirigidas por ADN/metabolismo , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Genoma Viral/genética , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Pandemias , Conformación Proteica en Hélice alfa , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/metabolismo
14.
Nucleic Acids Res ; 48(11): e64, 2020 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-32352514

RESUMEN

The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Ensayos Analíticos de Alto Rendimiento , ARN Guia/genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Conjuntos de Datos como Asunto , Modelos Lineales , Reproducibilidad de los Resultados
15.
Nat Commun ; 11(1): 2422, 2020 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-32415118

RESUMEN

Transcription is fundamentally noisy, leading to significant heterogeneity across bacterial populations. Noise is often attributed to burstiness, but the underlying mechanisms and their dependence on the mode of promotor regulation remain unclear. Here, we measure E. coli single cell mRNA levels for two stress responses that depend on bacterial sigma factors with different mode of transcription initiation (σ70 and σ54). By fitting a stochastic model to the observed mRNA distributions, we show that the transition from low to high expression of the σ70-controlled stress response is regulated via the burst size, while that of the σ54-controlled stress response is regulated via the burst frequency. Therefore, transcription initiation involving σ54 differs from other bacterial systems, and yields bursting kinetics characteristic of eukaryotic systems.


Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Adenosina Trifosfato/química , Teorema de Bayes , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrólisis , Cinética , Modelos Genéticos , Regiones Promotoras Genéticas , ARN Polimerasa Sigma 54/metabolismo , ARN Mensajero/metabolismo , Factor sigma/metabolismo , Análisis de la Célula Individual , Procesos Estocásticos
16.
Nat Commun ; 11(1): 2465, 2020 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-32424289

RESUMEN

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma of B-cell origin with frequent expression of functional B-cell receptors (BCRs). Here we report that expression cloning followed by antigen screening identifies DNA-directed RNA polymerase beta' (RpoC) from Moraxella catarrhalis as frequent antigen of BCRs of IgD+ LP cells. Patients show predominance of HLA-DRB1*04/07 and the IgVH genes encode extraordinarily long CDR3s. High-titer, light-chain-restricted anti-RpoC IgG1/κ-type serum-antibodies are additionally found in these patients. RpoC and MID/hag, a superantigen co-expressed by Moraxella catarrhalis that is known to activate IgD+ B cells by binding to the Fc domain of IgD, have additive activation effects on the BCR, the NF-κB pathway and the proliferation of IgD+ DEV cells expressing RpoC-specific BCRs. This suggests an additive antigenic and superantigenic stimulation of B cells with RpoC-specific IgD+ BCRs under conditions of a permissive MHC-II haplotype as a model of NLPHL lymphomagenesis, implying future treatment strategies.


Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos B/inmunología , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/microbiología , Moraxella catarrhalis/inmunología , Adolescente , Adulto , Anciano , Autoantígenos/inmunología , Línea Celular Tumoral , Proliferación Celular , Niño , ARN Polimerasas Dirigidas por ADN/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Enfermedad de Hodgkin/sangre , Humanos , Inmunoglobulina D/metabolismo , Fragmentos Fab de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Masculino , Persona de Mediana Edad , Modelos Biológicos , Receptores de Antígenos de Linfocitos B/metabolismo
17.
PLoS One ; 15(5): e0232927, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32396566

RESUMEN

Tetraene macrolides remain one of the most reliable fungicidal agents as resistance of fungal pathogens to these antibiotics is relatively rare. The modes of action and biosynthesis of polyene macrolides had been the focus of research over the past few years. However, few studies have been carried out on the overproduction of polyene macrolides. In the present study, cumulative drug-resistance mutation was used to obtain a quintuple mutant G5-59 with huge tetraene macrolide overproduction from the starting strain Streptomyces diastatochromogenes 1628. Through DNA sequence analysis, the mutation points in the genes of rsmG, rpsL and rpoB were identified. Additionally, the growth characteristic and expression level of tetrRI gene (belonging to the large ATP binding regulator of LuxR family) involved in the biosynthesis of tetraene macrolides were analyzed. As examined with 5L fermentor, the quintuple mutant G5-59 grew very well and the maximum productivity of tetramycin A, tetramycin P and tetrin B was as high as 1735, 2811 and 1500 mg/L, which was 8.7-, 16- and 25-fold higher than that of the wild-type strain 1628, respectively. The quintuple mutant G5-59 could be useful for further improvement of tetraene macrolides production at industrial level.


Asunto(s)
Proteínas Bacterianas/genética , Reactores Biológicos/microbiología , Macrólidos/metabolismo , Mutación , Streptomyces/crecimiento & desarrollo , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana , Fermentación , Ingeniería Metabólica , Metiltransferasas/genética , Proteínas Ribosómicas/genética , Análisis de Secuencia de ADN , Streptomyces/genética , Streptomyces/metabolismo
18.
Nature ; 582(7813): 561-565, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32365353

RESUMEN

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.


Asunto(s)
Betacoronavirus/genética , Clonación Molecular/métodos , Infecciones por Coronavirus/virología , Genoma Viral/genética , Genómica/métodos , Neumonía Viral/virología , Genética Inversa/métodos , Biología Sintética/métodos , Animales , China/epidemiología , Chlorocebus aethiops , Cromosomas Artificiales de Levadura/metabolismo , Infecciones por Coronavirus/epidemiología , ARN Polimerasas Dirigidas por ADN/metabolismo , Evolución Molecular , Humanos , Mutación , Pandemias/estadística & datos numéricos , Neumonía Viral/epidemiología , Virus Sincitiales Respiratorios/genética , Saccharomyces cerevisiae/genética , Células Vero , Proteínas Virales/metabolismo , Virus Zika/genética
19.
PLoS One ; 15(4): e0232332, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32353042

RESUMEN

The assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) is an inexpensive protocol for measuring open chromatin regions. ATAC-seq is also relatively simple and requires fewer cells than many other high-throughput sequencing protocols. Therefore, it is tractable in numerous settings where other high throughput assays are challenging to impossible. Hence it is important to understand the limits of what can be inferred from ATAC-seq data. In this work, we leverage ATAC-seq to predict the presence of nascent transcription. Nascent transcription assays are the current gold standard for identifying regions of active transcription, including markers for functional transcription factor (TF) binding. We combine mapped short reads from ATAC-seq with the underlying peak sequence, to determine regions of active transcription genome-wide. We show that a hybrid signal/sequence representation classified using recurrent neural networks (RNNs) can identify these regions across different cell types.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Análisis de Secuencia de ADN/métodos , Sitio de Iniciación de la Transcripción , Células A549 , Células HCT116 , Humanos , Células MCF-7 , Redes Neurales de la Computación , Motivos de Nucleótidos , Unión Proteica , Factores de Transcripción/metabolismo
20.
Cells ; 9(5)2020 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-32443810

RESUMEN

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Neumonía Viral/virología , ARN Viral/metabolismo , Replicación Viral/fisiología , Animales , Dominio Catalítico , ARN Polimerasas Dirigidas por ADN/metabolismo , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Genoma Viral/genética , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Pandemias , Conformación Proteica en Hélice alfa , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/metabolismo
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