RESUMEN
Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.
Asunto(s)
Glutaminasa , Glutamina , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Ligasas de Carbono-Nitrógeno , Microscopía por Crioelectrón , Citidina Trifosfato/química , Glutaminasa/química , Glutaminasa/metabolismo , Glutamina/metabolismo , Guanosina Trifosfato/química , Óxido Nítrico Sintasa/metabolismo , Uridina Trifosfato/química , Uridina Trifosfato/metabolismoRESUMEN
Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5'- or 3'-end of the molecule. An unsurpassed method for 5'-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ-32P]ATP. In the case of 3'-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3'-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3'-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e. [5'-32P]pCp ligation to the RNA 3'-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.
Asunto(s)
Nucleotidiltransferasas/química , ARN/química , Coloración y Etiquetado/métodos , Citidina Trifosfato/química , Técnicas In Vitro , Marcaje Isotópico/métodos , Nucleótidos/química , Radioisótopos de Fósforo , ARN/genética , ARN Ligasa (ATP)/química , Coloración y Etiquetado/normas , Especificidad por Sustrato , Uridina Trifosfato/químicaRESUMEN
Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Edición de ARN , Trypanosoma brucei brucei/genética , Fluoresceína/química , Colorantes Fluorescentes/química , Genoma Mitocondrial , Reacción en Cadena de la Polimerasa Multiplex/métodos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Uridina Trifosfato/químicaRESUMEN
AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/química , Betacoronavirus/enzimología , Infecciones por Coronavirus/tratamiento farmacológico , Guanosina Monofosfato/análogos & derivados , Neumonía Viral/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Ribavirina/química , Sofosbuvir/química , Proteínas Virales/antagonistas & inhibidores , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Alanina/química , Alanina/metabolismo , Alphacoronavirus/enzimología , Alphacoronavirus/genética , Secuencia de Aminoácidos , Antivirales/metabolismo , Betacoronavirus/genética , COVID-19 , Dominio Catalítico , Biología Computacional/métodos , Infecciones por Coronavirus/virología , Reposicionamiento de Medicamentos/métodos , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Neumonía Viral/virología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Ribavirina/metabolismo , SARS-CoV-2 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sofosbuvir/metabolismo , Termodinámica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies, we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical-SAM enzymes. We predict that our work will help in using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues.
Asunto(s)
Citidina Trifosfato/química , Proteínas Fúngicas/química , Proteínas/química , S-Adenosilmetionina/química , Sordariales/química , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Citidina Trifosfato/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Sordariales/clasificación , Sordariales/enzimología , Homología Estructural de Proteína , Especificidad por Sustrato , Termodinámica , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/química , Uridina Trifosfato/metabolismoRESUMEN
Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak-trigger-freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.
Asunto(s)
Ligasas de Carbono-Nitrógeno/química , ARN Polimerasas Dirigidas por ADN/química , ARN Bacteriano/química , Transcripción Genética , Bacillus subtilis/química , Bacillus subtilis/genética , Ligasas de Carbono-Nitrógeno/genética , Cristalografía por Rayos X , ADN/química , ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Factor sigma/química , Factor sigma/genética , Uridina Trifosfato/química , Uridina Trifosfato/genéticaRESUMEN
Viperin is a radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by additional undefined mechanisms operating through its N- and C-terminal domains. Here, we describe crystal structures of viperin bound to a SAM analogue and CTP or uridine triphosphate (UTP) and report kinetic parameters for viperin-catalyzed reactions with CTP or UTP as substrates. Viperin orients the C4' hydrogen atom of CTP and UTP similarly for abstraction by a 5'-deoxyadenosyl radical, but the uracil moiety introduces unfavorable interactions that prevent tight binding of UTP. Consistently, kcat is similar for CTP and UTP whereas the Km for UTP is much greater. The structures also show that nucleotide binding results in ordering of the C-terminal tail and reveal that this region contains a P-loop that binds the γ-phosphate of the bound nucleotide. Collectively, the results explain the selectivity for CTP and reveal a structural role for the C-terminal tail in binding CTP and UTP.
Asunto(s)
Citidina Trifosfato/química , Proteínas/química , Proteínas/metabolismo , S-Adenosilhomocisteína/química , Uridina Trifosfato/química , Animales , Cristalografía por Rayos X , Citidina Trifosfato/metabolismo , Cinética , Ratones , Modelos Moleculares , Estructura Molecular , Mutación , Proteínas/genética , S-Adenosilhomocisteína/metabolismo , Especificidad por Sustrato , Uridina Trifosfato/metabolismoRESUMEN
The nucleotide receptors P2Y2 and P2Y4 are the most closely related G protein-coupled receptors (GPCRs) of the P2Y receptor (P2YR) family. Both subtypes couple to Gq proteins and are activated by the pyrimidine nucleotide UTP, but only P2Y2R is also activated by the purine nucleotide ATP. Agonists and antagonists of both receptor subtypes have potential as drugs e.g. for neurodegenerative and inflammatory diseases. So far, potent and selective, "drug-like" ligands for both receptors are scarce, but would be required for target validation and as lead structures for drug development. Structural information on the receptors is lacking since no X-ray structures or cryo-electron microscopy images are available. Thus, we performed receptor homology modeling and docking studies combined with mutagenesis experiments on both receptors to address the question how ligand binding selectivity for these closely related P2YR subtypes can be achieved. The orthosteric binding site of P2Y2R appeared to be more spacious than that of P2Y4R. Mutation of Y197 to alanine in P2Y4R resulted in a gain of ATP sensitivity. Anthraquinone-derived antagonists are likely to bind to the orthosteric or an allosteric site depending on their substitution pattern and the nature of the orthosteric binding site of the respective P2YR subtype. These insights into the architecture of P2Y2- and P2Y4Rs and their interactions with structurally diverse agonists and antagonist provide a solid basis for the future design of potent and selective ligands.
Asunto(s)
Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2/metabolismo , Sitios de Unión/genética , Línea Celular Tumoral , Microscopía por Crioelectrón/métodos , Desarrollo de Medicamentos , Humanos , Ligandos , Modelos Moleculares , Mutagénesis/genética , Nucleótidos/química , Nucleótidos/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/fisiología , Transducción de Señal/genética , Relación Estructura-Actividad , Uridina Trifosfato/química , Uridina Trifosfato/genéticaRESUMEN
Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Sondas de ADN/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Genómica/métodos , Hibridación Fluorescente in Situ/métodos , Técnicas Bacteriológicas/instrumentación , Técnicas Bacteriológicas/métodos , Técnicas de Cultivo de Célula/métodos , Línea Celular , Sondas de ADN/genética , ADN Bacteriano/genética , Nucleótidos de Desoxiuracil/química , Didesoxinucleótidos/química , Digoxigenina/análogos & derivados , Digoxigenina/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Secciones por Congelación , Genómica/instrumentación , Humanos , Hibridación Fluorescente in Situ/instrumentación , Rodaminas/química , Coloración y Etiquetado/instrumentación , Coloración y Etiquetado/métodos , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/químicaRESUMEN
A modified nucleoside triphosphate bearing two modifications based on a 2'-deoxy-2'-fluoro-arabinofuranose sugar and a uracil nucleobase equipped with a C5-ethynyl moiety (5-ethynyl-2'F-ANA UTP) was synthesized. This nucleotide analog could enzymatically be incorporated into DNA oligonucleotides by primer extension and reverse transcribed to unmodified DNA. This nucleotide could be used in SELEX for the identification of high binding affinity and nuclease resistant aptamers.
Asunto(s)
Aptámeros de Nucleótidos/química , Arabinosa/análogos & derivados , Uridina Trifosfato/química , Arabinosa/química , Sitios de Unión , Conformación de Carbohidratos , ADN/química , ADN/genéticaRESUMEN
RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.
Asunto(s)
Guanosina Trifosfato/química , Hepacivirus/enzimología , Simulación de Dinámica Molecular , ARN Polimerasa Dependiente del ARN/química , Uridina Trifosfato/química , Proteínas no Estructurales Virales/química , Secuencias de Aminoácidos , Dominio Catalítico , Guanosina Trifosfato/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Uridina Trifosfato/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
Nucleoside triphosphates (NTPs) are important synthetic targets with diverse applications in therapeutics and diagnostics. Enzymatic routes to NTPs from simple building blocks are attractive, however the cost and complexity of assembling the requisite mixtures of multiple enzymes hinders application. Here, we describe the use of an engineered E. coli cell-free lysate as an efficient readily-prepared multi-enzyme biocatalyst for the production of uridine triphosphate (UTP) from free ribose and nucleobase. Endogenous lysate enzymes are able to support the nucleobase ribosylation and nucleotide phosphorylation steps, while uridine phosphorylation and the production of ribose phosphates (ribose 1-phosphate, ribose 5-phosphate and phosphoribosyl pyrophosphate) require recombinant enrichment of endogenous activities. Co-expression vectors encoding all required recombinant enzymes were employed for host cell transformation, such that a cell-free lysate with all necessary activities was obtained from a single bacterial culture. ATP required as phosphorylation cofactor was recycled by endogenous lysate enzymes using cheap, readily-prepared acetyl phosphate. Surprisingly, acetyl phosphate initiated spontaneous generation of ATP in the lysate, most likely from the breakdown of endogenous pools of adenosine-containing starting materials (e.g. adenosine cofactors, ribonucleic acids). The sub-stoichiometric amount of ATP produced and recycled in this way was enough to support all ATP-dependent steps without addition of any exogenous cofactor or auxiliary enzyme. Using this approach, equimolar solutions of orotic acid and ribose are transformed near quantitatively into 1.4 g L-1 UTP within 2.5 h, using a low-cost, readily-generated biocatalytic preparation.
Asunto(s)
Adenosina Trifosfato/farmacología , Recombinación Genética , Ribosa/metabolismo , Uracilo/metabolismo , Uridina Trifosfato/biosíntesis , Catálisis , Escherichia coli/metabolismo , Hidrólisis , Ácido Orótico/metabolismo , Recombinación Genética/genética , Ribosa/química , Uracilo/química , Uridina Trifosfato/químicaRESUMEN
Expanding the chemical diversity of threose nucleic acid (TNA) beyond the natural bases would enable the development of TNA polymers with enhanced physicochemical properties. Here, we describe a versatile approach for increasing the chemical diversity of TNA using 5-alkynyl-modified α-l-threofuranosyl uridine triphosphates that are substrates for a TNA polymerase.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Tetrosas/química , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/metabolismo , Especificidad por Sustrato , Uridina Trifosfato/químicaRESUMEN
This unit describes a simple, reliable, and efficient chemical method for the synthesis of 5-(3-aminoallyl)-2'-deoxyuridine-5'-O-triphosphate (AA-dUTP) and 5-(3-aminoallyl)-uridine-5'-O-triphosphate (AA-UTP), starting from the corresponding nucleoside triphosphate. The presented strategy involves regioselective iodination of nucleoside triphosphate using N-iodosuccinimide followed by the palladium-catalyzed Heck coupling with allylamine to provide the corresponding (E)-5-aminoallyl-uridine-5'-O-triphosphate in good yields. It is noteworthy that the protocol not only provides a high-purity product but also eliminates the use of toxic mercuric reagents. © 2017 by John Wiley & Sons, Inc.
Asunto(s)
Nucleótidos/química , Paladio/química , Uridina Trifosfato/síntesis química , Catálisis , Yodo/química , Uridina Trifosfato/químicaRESUMEN
6-O-(2-Nitrobenzyl)guanosine and 4-O-(2-nitrobenzyl)uridine triphosphates (NBGTP, NBUTP) were synthesized, and their biochemical and photophysical properties were evaluated. We synthesized NBUTP using the canonical triphosphate synthesis method and NBGTP from 2',3'-O-TBDMS guanosine via a triphosphate synthesis method by utilizing mild acidic desilylation conditions. Deprotection of the nitrobenzyl group in NBGTP and NBUTP proceeded within 60s by UV irradiation at 365nm. Experiments using NBGTP or NBUTP in T7-RNA transcription reactions showed that NBGTP could be useful for the photocontrol of transcription by UV irradiation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Guanosina/farmacología , Transcripción Genética/efectos de los fármacos , Rayos Ultravioleta , Uridina Trifosfato/farmacología , Proteínas Virales/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Guanosina/análogos & derivados , Guanosina/síntesis química , Estructura Molecular , Relación Estructura-Actividad , Transcripción Genética/genética , Uridina Trifosfato/síntesis química , Uridina Trifosfato/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.
Asunto(s)
Dibenzocicloheptenos/síntesis química , Antagonistas del Receptor Purinérgico P2Y/síntesis química , Pirimidinonas/síntesis química , Receptores Purinérgicos P2Y2/metabolismo , Uridina Trifosfato/química , Dibenzocicloheptenos/química , Dibenzocicloheptenos/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Unión Proteica , Antagonistas del Receptor Purinérgico P2Y/química , Antagonistas del Receptor Purinérgico P2Y/metabolismo , Pirimidinonas/química , Pirimidinonas/metabolismo , Receptores Purinérgicos P2Y2/química , Uridina Trifosfato/metabolismoRESUMEN
Although the genome is generally thought to be transcriptionally silent during mitosis, technical limitations have prevented sensitive mapping of transcription during mitosis and mitotic exit. Thus, the means by which the interphase expression pattern is transduced to daughter cells have been unclear. We used 5-ethynyluridine to pulse-label transcripts during mitosis and mitotic exit and found that many genes exhibit transcription during mitosis, as confirmed with fluorescein isothiocyanate-uridine 5'-triphosphate labeling, RNA fluorescence in situ hybridization, and quantitative reverse transcription polymerase chain reaction. The first round of transcription immediately after mitosis primarily activates genes involved in the growth and rebuilding of daughter cells, rather than cell type-specific functions. We propose that the cell's transcription pattern is largely retained at a low level through mitosis, whereas the amplitude of transcription observed in interphase is reestablished during mitotic exit.
Asunto(s)
Mitosis/genética , Transcripción Genética , Activación Transcripcional , Línea Celular Tumoral , Fluoresceína-5-Isotiocianato/química , Humanos , Hibridación Fluorescente in Situ , Interfase/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Mensajero/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y Etiquetado , Uridina Trifosfato/químicaRESUMEN
Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.
Asunto(s)
Proteínas Bacterianas/química , Erwinia amylovora/enzimología , Glucofosfatos/química , UTP-Glucosa-1-Fosfato Uridililtransferasa/química , Uridina Difosfato Glucosa/química , Uridina Trifosfato/química , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Erwinia amylovora/química , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosamina/análogos & derivados , Galactosamina/química , Galactosamina/metabolismo , Galactosafosfatos/química , Galactosafosfatos/metabolismo , Expresión Génica , Glucosamina/análogos & derivados , Glucosamina/química , Glucosamina/metabolismo , Glucofosfatos/metabolismo , Cinética , Manosafosfatos/química , Manosafosfatos/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Pentosafosfatos/química , Pentosafosfatos/metabolismo , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/química , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3' end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3' end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.
Asunto(s)
Técnicas Genéticas , Saccharomycetales/genética , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al GTP/genética , Mutación , Regiones Promotoras Genéticas , ARN sin Sentido , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/química , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The development of modular and efficient methods to functionalize RNA with biophysical probes is very important in advancing the understanding of the structural and functional relevance of RNA in various cellular events. Herein, we demonstrate a two-step bioorthogonal chemical functionalization approach for the conjugation of multiple probes onto RNA transcripts using a 5-vinyl-modified uridine nucleotide analog (VUTP). VUTP, containing a structurally noninvasive and versatile chemoselective handle, was efficiently incorporated into RNA transcripts by in vitro transcription reactions. Furthermore, we show for the first time the use of a palladium-mediated oxidative Heck reaction in functionalizing RNA with fluorogenic probes by reacting vinyl-labeled RNA transcripts with appropriate boronic acid substrates. The vinyl label also permitted the post-transcriptional functionalization of RNA by a reagent-free inverse electron demand Diels-Alder (IEDDA) reaction in the presence of tetrazine substrates. Collectively, our results demonstrate that the incorporation of VUTP provides newer possibilities for the modular functionalization of RNA with variety of reporters.