RESUMEN
In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0-10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.
Asunto(s)
Aptámeros de Nucleótidos , MicroARNs , Proteínas Virales , MicroARNs/análisis , MicroARNs/sangre , Humanos , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Límite de Detección , Conformación de Ácido Nucleico , Espectrometría de Fluorescencia , ARN Polimerasas Dirigidas por ADN/químicaRESUMEN
The limited therapeutic options and increasing drug-resistance call for next-generation influenza antivirals. Due to the essential function in viral replication and high sequence conservation among influenza viruses, influenza polymerase PA-PB1 protein-protein interaction becomes an attractive drug target. Here, we developed an in vitro split luciferase complementation-based assay to speed up screening of PA-PB1 interaction inhibitors. By screening 10,000 compounds, we identified two PA-PB1 interaction inhibitors, R160792 and R151785, with potent and broad-spectrum antiviral activity against a panel of influenza A and B viruses, including amantadine-, oseltamivir-, or dual resistant strains. Further mechanistic study reveals that R151785 inhibits PA nuclear localization, reduces the levels of viral RNAs and proteins, and inhibits viral replication at the intermediate stage, all of which are in line with its antiviral mechanism of action. Overall, we developed a robust high throughput-screening assay for screening broad-spectrum influenza antivirals targeting PA-PB1 interaction and identified R151785 as a promising antiviral drug candidate.
Asunto(s)
Antivirales/química , ARN Polimerasas Dirigidas por ADN/química , Luciferasas/química , Inhibidores de la Síntesis del Ácido Nucleico/química , Orthomyxoviridae/enzimología , Proteínas Virales/química , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
In this work, we achieved the selective detection of wild and mutated rpoB gene in M. tuberculosis using an electrochemical DNA (E-DNA) sensor based on polypyrrole/Fe3O4 nanocomposite bearing redox naphthoquinone tag on PAMAM (spaNQ/PAMAM/PPy/Fe3O4). The hybridization between a given probe and the complementary DNA target induced a large decrease in the naphthoquinone redox signal as measured by SWV and no cross-hybridization with single nucleotide mismatch DNA target occurred. Thanks to the catalytic properties of iron oxide nanoparticles combined with conducting properties of polypyrrole platform, we demonstrated that the transducing system allowed the detection of 1 fM of DNA target in a 50-µL drop corresponding to 3â¯×â¯104 copies of DNA. The sensor was able to detect the rpoB gene in PCR-amplified samples of genomic DNA and could also discriminate between the wild type rpoB gene and a single nucleotide mutated rpoB gene that provides resistance to rifampicin. Furthermore, the sensor could selectively detect the wild and mutant DNA in genomic samples without PCR amplification.
Asunto(s)
Proteínas Bacterianas/genética , Técnicas Biosensibles , ADN Bacteriano/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/genética , Mycobacterium tuberculosis/aislamiento & purificación , Proteínas Bacterianas/química , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/química , Compuestos Férricos/química , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Nanocompuestos/química , Polímeros/química , Pirroles/químicaRESUMEN
Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5'triphosphate-3'diphosphate and guanosine 3', 5'-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5' and 3' moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic. Here, we use (p)ppGpp's adenine analogs [(p)ppApp] to investigate how the nucleobase contributes to determine its binding site and transcriptional regulation. We determined X-ray crystal structure of Escherichia coli RNAP-(p)ppApp complex, which shows the analogs bind near the active site and switch regions of RNAP. We have also explored the regulatory effects of (p)ppApp on transcription initiating from the well-studied E. coli rrnB P1 promoter to assess and compare properties of (p)ppApp with (p)ppGpp. We demonstrate that contrary to (p)ppGpp, (p)ppApp activates transcription at this promoter and DksA hinders this effect. Moreover, pppApp exerts a stronger effect than ppApp. We also show that when ppGpp and pppApp are present together, the outcome depends on which one of them was pre-incubated with RNAP first. This behavior suggests a surprising Yin-Yang like reciprocal plasticity of RNAP responses at a single promoter, occasioned simply by pre-exposure to one or the other nucleotide. Our observations underscore the importance of the (p)ppNpp's purine nucleobase for interactions with RNAP, which may lead to a better fundamental understanding of (p)ppGpp regulation of RNAP activity.
Asunto(s)
Nucleótidos de Adenina/química , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Activación Transcripcional , Nucleótidos de Adenina/metabolismo , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Guanosina Pentafosfato/química , Guanosina Pentafosfato/metabolismo , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Structure-based pharmacophores were generated and validated using the bioactive conformations of different co-crystallized enzyme-inhibitor complexes for allosteric palm-1 and thumb-2 inhibitors of NS5B. Two pharmacophore models were obtained, one for palm-1 inhibitors with sensitivity = 0.929 and specificity = 0.983, and the other for thumb-2 inhibitors with sensitivity = 1 and specificity = 0.979. In addition, a quantitative structure activity relationship (QSAR) models were developed based on using the values of different scoring functions as descriptors predicting the activity on both allosteric binding sites (palm-1 and thumb-2). QSAR studies revealed good predictive and statistically significant two descriptor models (r2 = .837, r2adjusted = .792 and r2prediction = .688 for palm-1 model and r2 = .927, r2adjusted = .908 and r2prediction = .779 for thumb-2 model). External validation for the QSAR models assured their prediction power with r2ext = .72 and .89 for palm-1 and thumb-2, respectively. Different docking protocols were examined for their validity to predict the correct binding poses of inhibitors inside their respective binding sites. Virtual screening was carried out on ZINC database using the generated pharmacophores, the selected valid docking algorithms and QSAR models to find compounds that could theoretically bind to both sites simultaneously.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepatitis C/genética , Regulación Alostérica/genética , Sitio Alostérico , Antivirales/química , Antivirales/uso terapéutico , ARN Polimerasas Dirigidas por ADN/química , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Hepacivirus/genética , Hepacivirus/patogenicidad , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica/efectos de los fármacos , Relación Estructura-Actividad Cuantitativa , ARN Viral/efectos de los fármacos , ARN Viral/genéticaRESUMEN
The incidence of bacterial disease has increased tremendously in the last decade, because of the emergence of drug resistance strains within the bacterial pathogens. The present study was to investigate the antibacterial compound 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ) isolated from marine Streptomyces sp. VITVSK1 as a potent antibacterial agent. The antibacterial potential of DTBBQ was investigated against RNA Polymerase (PDB ID-1I6V) by in silico molecular docking tools. Results of our study showed the high affinity interaction between DTBBQ and RNA polymerase and also confirmed the drug likeliness of DTBBQ using ADMET in silico pharmacology tools. Our findings suggest that DTBBQ could be used as antibacterial drug to defend the emerging antibacterial resistance.
Asunto(s)
Antibacterianos/química , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/ultraestructura , Simulación del Acoplamiento Molecular/métodos , Streptomyces/química , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática , Modelos Químicos , Conformación Molecular , Unión Proteica , Especificidad de la EspecieRESUMEN
The identification of a novel hit compound inhibitor of the protein-protein interaction between the influenza RNA-polymerase PA and PB1 subunits has been accomplished by means of high-throughput screening. A small family of structurally related molecules has been synthesized and biologically evaluated with most of the compounds showing micromolar potency of inhibition against viral replication.
Asunto(s)
Antivirales/toxicidad , Benzoxazoles/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Inhibidores Enzimáticos/síntesis química , Virus de la Influenza A/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Benzoxazoles/síntesis química , Benzoxazoles/toxicidad , ARN Polimerasas Dirigidas por ADN/química , Perros , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Virus de la Influenza A/enzimología , Células de Riñón Canino Madin Darby , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-ActividadRESUMEN
In order to find new molecules for antiviral drug design, we screened 102 ethyl acetate extracts from New-Caledonian flora for antiviral activity against the dengue 2 virus RNA-dependant RNA polymerase (DV-NS5 RdRp). The leaf extract of Dacrydium balansae, which strongly inhibited the DV-NS5, was submitted to bioguided fractionation. Four biflavonoids ( 1- 4), three sterols ( 5- 7), and two stilbene derivatives ( 8- 9) were identified and evaluated for their antiviral potential on the DV-NS5 RdRp. Biflavonoids appeared to be potent inhibitors of DV-NS5 RdRp with IC (50)s between 0.26 and 3.12 µM. Inhibitory activity evaluations against the RNA polymerase from other Flaviviridae viruses allowed us to conclude that these compounds are specific inhibitors of the DV RNA polymerase. The strongest inhibitions were observed with hinokiflavone ( 4), but podocarpusflavone A ( 2) is the strongest noncytotoxic inhibitor of the DV-NS5 and it also displayed polymerase inhibitory activity in a DV replicon. A preliminary structure-activity relationship study (SARs) revealed the necessity of the biflavonoid skeleton, the influence of number and position of methoxylations, and the importance of a free rotation of the linkage between the two apigenin monomers of the biflavonoids. To the best of our knowledge, podocarpusflavone A ( 2) is the strongest noncytotoxic non-nucleotide molecule exhibiting a specific inhibitory activity against the RNA polymerase domain of DV-NS5 and thus is promising for chemotherapy development against dengue fever.
Asunto(s)
Antivirales/farmacología , Biflavonoides/farmacología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Virus del Dengue/enzimología , Inhibidores Enzimáticos/farmacología , Fitoterapia , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Tracheophyta/química , Antivirales/química , Biflavonoides/química , ARN Polimerasas Dirigidas por ADN/química , Virus del Dengue/efectos de los fármacos , Nueva Caledonia , Corteza de la Planta/química , Hojas de la Planta/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
OBJECTIVES: Hepatitis C virus (HCV) infection causes chronic liver disease and is a major public health problem worldwide. The aim of this study was to evaluate the potential of Monascus pigment derivatives, which were derived from a microbial secondary metabolite synthesized from polyketides by Monascus spp., as HCV antiviral agents. METHODS: We performed an in vitro RNA-dependent RNA polymerase (RdRp) assay to screen for HCV RdRp inhibitors. The anti-HCV activity of RdRp inhibitors in HCV-replicating cells was evaluated by quantification of the RNA viral genome. Molecular docking analysis was performed to predict the binding sites of the selected RdRp inhibitors. RESULTS: We have identified a Monascus pigment and its derivatives as inhibitors of the HCV NS5B RdRp. A group of Monascus orange pigment (MOP) amino acid derivatives, in which the reactive oxygen moiety was changed to amino acids, significantly inhibited HCV replication. Further, combination of the MOP derivatives (Phe, Val or Leu conjugates) with interferon (IFN)-α inhibited HCV replication more than IFN-α treatment alone. Lastly, molecular docking studies indicate the inhibitors may bind to a thumb subdomain allosteric site of NS5B. The antiviral activity of the MOP derivatives was related to a modulation of the mevalonate pathway, since the mevalonate-induced increase in HCV replication was suppressed by the MOP compounds. CONCLUSIONS: Our results identify amino acid derivatives of MOP as potential anti-HCV agents and suggest that their combination with IFN-α might offer an alternative strategy for the control of HCV replication.
Asunto(s)
Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Hepacivirus/efectos de los fármacos , Ácido Mevalónico/metabolismo , Monascus/química , Pigmentos Biológicos/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/química , Antivirales/aislamiento & purificación , Sitios de Unión , Vías Biosintéticas/genética , ARN Polimerasas Dirigidas por ADN/química , Evaluación Preclínica de Medicamentos/métodos , Humanos , Simulación de Dinámica Molecular , Pigmentos Biológicos/química , Pigmentos Biológicos/aislamiento & purificaciónRESUMEN
The small regulatory protein Crl binds to sigmaS, the RNA polymerase stationary phase sigma factor. Crl facilitates the formation of the sigmaS-associated holoenzyme (EsigmaS) and thereby activates sigmaS-dependent genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with sigmaS, which results in an increase of the association rate of sigmaS to core RNA polymerase without any effect on the dissociation rate of EsigmaS. Crl is also able to associate with preformed EsigmaS with a higher affinity than with sigmaS alone. Furthermore, even at saturating sigmaS concentrations, Crl significantly increases EsigmaS association with the katN promoter and the productive isomerization of the EsigmaS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to sigma70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the sigma70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-sigmaS holoenzymes.
Asunto(s)
Proteínas Bacterianas/química , ARN Polimerasas Dirigidas por ADN/química , Regiones Promotoras Genéticas/fisiología , Salmonella enterica/química , Factor sigma/química , Factores de Transcripción/química , Transcripción Genética/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catalasa/química , Catalasa/genética , Catalasa/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Unión Proteica/fisiología , Salmonella enterica/genética , Salmonella enterica/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Resonancia por Plasmón de Superficie , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Both transcription and replication of the influenza virus RNA genome are catalysed by a virus-specific RNA polymerase. Recently, an in vitro assay, based on the synthesis of pppApG, for the initiation of replication by recombinant RNA polymerase in the absence of added primer was described. Here, these findings are extended to show that adenosine, AMP and ADP can each substitute for ATP in reactions catalysed by either recombinant ribonucleoprotein or RNA polymerase complexes with either model virion RNA (vRNA) or cRNA promoters. The use of either adenosine or AMP, rather than ATP, provides a convenient, sensitive and easy assay of replication initiation. Moreover, no pppApG was detected when a PB1-PA dimer, rather than the trimeric polymerase, was used to catalyse synthesis, contrasting with a previous report using baculovirus-expressed influenza RNA polymerase. Overall, it is suggested that the heterotrimeric polymerase is essential for the initiation of replication.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Orthomyxoviridae/fisiología , Adenosina/fisiología , Adenosina Difosfato , Adenosina Trifosfato/fisiología , Línea Celular , ARN Polimerasas Dirigidas por ADN/química , Dimerización , Humanos , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Subunidades de Proteína/metabolismo , ARN Complementario/genética , ARN Complementario/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
SAD phasing has been revisited recently, with experiments being carried out using previously unconventional sources of anomalous signal, particularly lighter atoms and softer X-rays. A case study is reported using the 75 kDa RNA-dependent RNA polymerase of the bacteriophase phi6, which binds a Mn atom and crystallizes with three molecules in the asymmetric unit. X-ray diffraction data were collected at a wavelength of 1.89 A and although the calculated anomalous signal from the three Mn atoms was only 1.2%, SHELXD and SOLVE were able to locate these atoms. SOLVE/RESOLVE used this information to obtain SAD phases and automatically build a model for the core region of the protein, which possessed the characteristic features of the right-hand polymerase motif. These results demonstrate that with modern synchrotron beamlines and software, manganese phasing is a practical tool for solving the structure of large proteins.
Asunto(s)
Cristalografía por Rayos X/métodos , Manganeso/química , Secuencias de Aminoácidos , Automatización , Bacteriófago phi 6/química , Calcio/química , Cationes , Cristalografía , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/metabolismo , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Proteínas/química , Programas Informáticos , Difracción de Rayos X , Rayos XRESUMEN
The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.
Asunto(s)
Bacterias/enzimología , Biopolímeros/biosíntesis , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/metabolismo , ARN/biosíntesis , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Bacterias/genética , Sitios de Unión , Biopolímeros/genética , Biopolímeros/metabolismo , Cristalización , Cristalografía por Rayos X , Citidina Trifosfato/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Geobacillus stearothermophilus/enzimología , Modelos Moleculares , Conformación Proteica , ARN/genética , ARN/metabolismo , Relación Estructura-Actividad , Moldes GenéticosRESUMEN
The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase alpha subunit C-terminal domain. Using preparations of E. coli RNA polymerase holoenzyme, specifically labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase alpha subunit in transcriptionally competent complexes at a class II FNR-dependent promoter. We conclude that one alpha subunit C-terminal domain binds immediately upstream of FNR, and that its position and orientation is the same as at similar promoters dependent on CRP, another E. coli transcription activator that is related to FNR. In complementary experiments, we show that the second alpha subunit C-terminal domain of RNA polymerase can be repositioned by upstream-bound CRP, but not by upstream-bound FNR.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Ácido Edético/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Hierro-Azufre/metabolismo , Regiones Promotoras Genéticas , Secuencia de Bases , Sitios de Unión , Proteína Receptora de AMP Cíclico/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Dimerización , Ácido Edético/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transactivadores , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K(D) in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.
Asunto(s)
Kinetoplastida/metabolismo , Edición de ARN , ARN/química , Animales , Secuencia de Bases , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/química , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligonucleótidos/química , ARN Mensajero/química , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Termodinámica , Trypanosoma brucei brucei/metabolismo , Proteínas Virales/químicaRESUMEN
The high affinity and specificity of nucleic acid base complementarity has been proven to be a powerful method for constructing specific molecular assemblies. On the other hand, recent structural studies of RNA have revealed the wide range of tertiary interactions utilized in RNA folding, which may potentially be used as tools for the design of specific macromolecular assemblies. Here, RNA building blocks containing two hairpin loops, based on the dimerization initiation site (DIS) of HIV RNA, connected by a short linker were used to construct large RNA assemblies through hairpin loop-loop ("kissing") interactions. We show that specific linear and circular assemblies can be constructed in a magnesium-dependent manner using several non-self-complementary loop-loop interactions designed in this study. These results show that the use of RNA tertiary interactions may broaden the repertoire of nucleic acid-based nanostructures.
Asunto(s)
Magnesio/química , ARN/química , Dicroismo Circular , ARN Polimerasas Dirigidas por ADN/química , Dimerización , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , VIH-1/química , Conformación de Ácido Nucleico , ARN/síntesis química , ARN Viral/químicaRESUMEN
Promoter opening, in which the complementary DNA strands separate around the transcriptional start site, is generally thermoreversible. An exceptional case of thermoirreversible opening of the T4 late promoter has been analyzed by KMnO4 footprinting and transcription. T4 late promoters, which consist of an 8-base pair (bp) TATA box "-10" element, are recognized by the small, phage-encoded, highly diverged sigma-family initiation subunit gp55. The T4 late promoter only opens above 15-20 degrees C, but once it has been formed remains open and transcriptionally active for days at -0.5 degrees C. The low temperature-trapped open complex and its isothermally formed state are shown to be structurally distinctive. Two "extended -10" sigma 70 promoters, which, like the T4 late promoter, lack "-35" sites, have been subjected to a comparative analysis: the T4 middle promoter PrIIB2 opens and closes thermoreversibly under conditions of basal and MotA- and AsiA-activated transcription. The open galP1 promoter complex, whose transcription bubble is very AT-rich, also closes reversibly upon shift to -0.5 degrees C, but more slowly than does the rIIB2 promoter. Formation of a trapped-open low temperature state of the promoter complex appears to be a singular property of gp55-RNA polymerase holoenzyme.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/enzimología , Regiones Promotoras Genéticas , Factor sigma/química , Proteínas Virales , Secuencia de Bases , ADN/metabolismo , Heparina/metabolismo , Calor , Cinética , Modelos Químicos , Datos de Secuencia Molecular , Permanganato de Potasio/farmacología , Factor sigma/metabolismo , Temperatura , Factores de Tiempo , Transcripción GenéticaRESUMEN
We have crystallized, using several approaches that may be of general interest, T7 RNA polymerase (T7RP) and the T7 RNA polymerase-T7 lysozyme complex (T7RPL) in forms suitable for structure determination by X-ray crystallography. A series of polyhydric alcohols, sugars, amino and methylamino acids, compounds known to stabilize protein structure, were found to be critical for both crystallization and subsequent improvement of the crystal's diffraction resolution. Moreover, optimal crystallogenesis was achieved through an unconventional "reverse" vapor diffusion sitting drop method that is suitable for proteins that are insoluble at low ionic strength.T7RP has been crystallized in an orthorhombic form (I), space group P222, with cell parameters a=220 A, b=205 A, c=67 A and a monoclinic form (II), space group P21, with cell parameters a=229 A, b=205 A, c=70 A, beta=106 degrees. Crystal form I diffracts X-rays to 3.5 A and form II to 6.0 A. Three and six copies of the polymerase are predicted to be in the asymmetric unit forms I and II, respectively. Three monoclinic crystal forms of the T7RPL complex have been obtained in space group C2. Form I has cell parameters a=320 A, b=93 A, c=229 A, beta=129 degrees, form II has parameters a=293 A, b=93 A, c=68 A, beta=93 degrees, and form III has parameters a=270 A, b=93 A, c=63 A, beta=103 degrees. Crystal form I diffracts synchrotron wiggler radiation to 3.2 A and form III to 2.8 A. Calculations of crystal density imply three or four copies of the complex in form I and one copy in the asymmetric unit of forms II and III.
Asunto(s)
Bacteriófago T7/enzimología , Cristalografía por Rayos X/métodos , ARN Polimerasas Dirigidas por ADN/química , N-Acetil Muramoil-L-Alanina Amidasa/química , Aminoácidos , Betaína , Carbohidratos , Carnitina , Cristalización , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos , Metilaminas , N-Acetil Muramoil-L-Alanina Amidasa/farmacología , Alcoholes del Azúcar , Proteínas ViralesRESUMEN
The gene (POLR2L) encoding a 7.6-kDa subunit (hRPB7.6) of human RNA polymerase has been cloned. It compromises two exons, 116 and 227 bp, respectively, interspaced with an intron of about 2.1 kb. This gene, whose localization has been assigned to the short arm of chromosome 11 (position 11p15), is transcribed in HeLa cells as one major messenger RNA, which encodes a 67-residue polypeptide (7645 Da) that shares strong homologies with the corresponding subunits of other eukaryotic and archaeal RNA polymerase subunits. Like its yeast counterpart (ABC10 beta, encoded by the RPB10 gene), the hRPB7.6 subunit may be shared by all three classes of human nuclear RNA polymerase. Cysteine residues characteristic of an atypical zinc-binding domain are conserved in the homologous sequences of all six species analyzed. A small, related RNA polymerase subunit from vaccinia virus exhibits an identical set of cysteines, suggesting that these residues may be contribute to a crucial function in the multimeric RNA polymerases.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Genes , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 11/genética , Clonación Molecular , ADN Complementario/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/clasificación , Exones , Células HeLa , Humanos , Hibridación in Situ , Intrones , Datos de Secuencia Molecular , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Virus Vaccinia/enzimología , Virus Vaccinia/genéticaRESUMEN
Chloroplast and etioplast in vitro transcription systems from mustard have different functional properties, which is reflected in differences in phosphorylation status. Here we report another transcription control mechanism, which involves two plastid DNA-dependent RNA polymerases designated as peak A and peak B enzymes. Both are large multi-subunit complexes, but differ in their native molecular mass (> 700 kDa for peak A and ca. 420 kDa for peak B) and in their polypeptide composition. The A enzyme is composed of at least 13 polypeptides, while the B enzyme contains only four putative subunits. Peak B activity is inhibited by rifampicin, whereas that of peak A is resistant. RNA polymerase activity was compared for plastids from cotyledons of 4-day-old seedlings that were grown either under continuous light (chloroplasts) or in darkness (etioplasts), or were first dark-grown and then transferred to light for 16 h ('intermediate-type' plastids). While the total activity was approximately the same in all three cases, enzyme B was the predominant activity obtained from etioplasts and enzyme A that obtained from chloroplasts. Both had equal activity in preparations from the 'intermediate-type' plastid form. Both activation/inactivation and differential gene expression seem to play a role in the regulation of the plastid transcription machinery.