RESUMEN
La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3'-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3' protection activity.
Asunto(s)
Autoantígenos , Ribonucleoproteínas , Ribonucleoproteínas/metabolismo , Autoantígenos/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Poli A/metabolismo , ARN/genética , ARN/metabolismo , Unión ProteicaRESUMEN
T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Motivated by our previous finding that intron retention (IR) could lead to transcript instability, in this study, we performed BruChase-Seq to experimentally monitor the expression dynamics of nascent transcripts in resting and activated CD4+ T cells. Computational modeling was then applied to quantify the stability of spliced and intron-retained transcripts on a genome-wide scale. Beyond substantiating that intron-retained transcripts were considerably less stable than spliced transcripts, we found a global stabilization of spliced mRNAs upon T cell activation, although the stability of intron-retained transcripts remained relatively constant. In addition, we identified that La-related protein 4 (LARP4), an RNA-binding protein (RBP) known to enhance mRNA stability, was involved in T cell activation-dependent mRNA stabilization. Knocking out Larp4 in mice destabilized Nfκb1 mRNAs and reduced secretion of interleukin-2 (IL2) and interferon-gamma (IFNγ), two factors critical for T cell proliferation and function. We propose that coordination between splicing regulation and mRNA stability may provide a novel paradigm to control spatiotemporal gene expression during T cell activation.
Asunto(s)
Interferón gamma/genética , Interleucina-2/genética , Proteínas/genética , Estabilidad del ARN/genética , Transcriptoma/genética , Empalme Alternativo/genética , Animales , Humanos , Intrones/genética , Activación de Linfocitos/genética , Ratones , FN-kappa B/genética , Unión Proteica/genética , ARN Mensajero/genética , Linfocitos T/metabolismoRESUMEN
The protein domain arrangement known as the La-module, comprised of a La motif (LaM) followed by a linker and RNA recognition motif (RRM), is found in seven La-related proteins: LARP1, LARP1B, LARP3 (La protein), LARP4, LARP4B, LARP6, and LARP7 in humans. Several LARPs have been characterized for their distinct activity in a specific aspect of RNA metabolism. The La-modules vary among the LARPs in linker length and RRM subtype. The La-modules of La protein and LARP7 bind and protect nuclear RNAs with UUU-3' tails from degradation by 3' exonucleases. LARP4 is an mRNA poly(A) stabilization factor that binds poly(A) and the cytoplasmic poly(A)-binding protein PABPC1 (also known as PABP). LARP1 exhibits poly(A) length protection and mRNA stabilization similar to LARP4. Here, we show that these LARP1 activities are mediated by its La-module and dependent on a PAM2 motif that binds PABP. The isolated La-module of LARP1 is sufficient for PABP-dependent poly(A) length protection and mRNA stabilization in HEK293 cells. A point mutation in the PAM2 motif in the La-module impairs mRNA stabilization and PABP binding in vivo but does not impair oligo(A) RNA binding by the purified recombinant La-module in vitro. We characterize the unusual PAM2 sequence of LARP1 and show it may differentially affect stable and unstable mRNAs. The unique LARP1 La-module can function as an autonomous factor to confer poly(A) protection and stabilization to heterologous mRNAs.
Asunto(s)
Autoantígenos/química , Autoantígenos/metabolismo , Oligopéptidos/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/metabolismo , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 9/agonistas , Sitios de Unión , Células HEK293 , Humanos , Motivos de Nucleótidos , Unión Proteica , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 9/metabolismo , Antígeno SS-BRESUMEN
Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of alpha-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.
Asunto(s)
ADN/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , ARN/metabolismo , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Secuencia de Aminoácidos , Animales , Emparejamiento Base , Cristalografía por Rayos X , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.7 microM, respectively. In contrast, the related tropolone analog beta-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 microM and 40 microM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both beta-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 microM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between beta-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.
Asunto(s)
Fármacos Anti-VIH/farmacología , Benzocicloheptenos/farmacología , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/antagonistas & inhibidores , Tropolona/análogos & derivados , Tropolona/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Benzocicloheptenos/química , Sitios de Unión , Cumarinas/metabolismo , Cumarinas/farmacología , VIH-1/enzimología , Piranocumarinas , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/metabolismo , Ribonucleasa H/metabolismo , Tropolona/químicaRESUMEN
Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.
Asunto(s)
ADN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Secuencia de Aminoácidos , Animales , Dimerización , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/enzimología , Humanos , Ratones , Datos de Secuencia Molecular , Poli A/metabolismo , Poli T/metabolismo , Estructura Terciaria de Proteína , ARN Bicatenario/metabolismo , Alineación de Secuencia , Resonancia por Plasmón de SuperficieRESUMEN
Human La antigen (Sjögren's syndrome antigen B [SSB]) is an abundant multifunctional RNA-binding protein. In the nucleoplasm, La binds to and protects from 3' exonucleases, the ends of precursor tRNAs, and other transcripts synthesized by RNA polymerase III and facilitates their maturation, while a nucleolar isoform has been implicated in rRNA biogenesis by multiple independent lines of evidence. We showed previously that conditional La knockout (La cKO) from mouse cortex neurons results in defective tRNA processing, although the pathway(s) involved in neuronal loss thereafter was unknown. Here, we demonstrate that La is stably associated with a spliced pre-tRNA intermediate. Microscopic evidence of aberrant nuclear accumulation of 5.8S rRNA in La cKO is supported by a 10-fold increase in a pre-5.8S rRNA intermediate. To identify pathways involved in subsequent neurodegeneration and loss of brain mass in the cKO cortex, we employed mRNA sequencing (mRNA-Seq), immunohistochemistry, and other approaches. This revealed robust enrichment of immune and astrocyte reactivity in La cKO cortex. Immunohistochemistry, including temporal analyses, demonstrated neurodegeneration, followed by astrocyte invasion associated with immune response and decreasing cKO cortex size over time. Thus, deletion of La from postmitotic neurons results in defective pre-tRNA and pre-rRNA processing and progressive neurodegeneration with loss of cortical brain mass.
Asunto(s)
Astrocitos/patología , Autoantígenos/genética , Gliosis/genética , Neuronas/patología , Precursores del ARN/metabolismo , ARN Ribosómico 5.8S/metabolismo , ARN de Transferencia/metabolismo , Ribonucleoproteínas/genética , Animales , Astrocitos/metabolismo , Autoantígenos/fisiología , Muerte Celular/genética , Células Cultivadas , Femenino , Gliosis/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Ribonucleoproteínas/fisiología , Antígeno SS-BRESUMEN
Messenger RNA function is controlled by the 3' poly(A) tail (PAT) and poly(A)-binding protein (PABP). La-related protein-4 (LARP4) binds poly(A) and PABP. LARP4 mRNA contains a translation-dependent, coding region determinant (CRD) of instability that limits its expression. Although the CRD comprises <10% of LARP4 codons, the mRNA levels vary >20 fold with synonymous CRD substitutions that accommodate tRNA dynamics. Separately, overexpression of the most limiting tRNA increases LARP4 levels and reveals its functional activity, net lengthening of the PATs of heterologous mRNAs with concomitant stabilization, including ribosomal protein (RP) mRNAs. Genetic deletion of cellular LARP4 decreases PAT length and RPmRNA stability. This LARP4 activity requires its PABP-interaction domain and the RNA-binding module which we show is sensitive to poly(A) 3'-termini, consistent with protection from deadenylation. The results indicate that LARP4 is a posttranscriptional regulator of ribosomal protein production in mammalian cells and suggest that this activity can be controlled by tRNA levels.
Asunto(s)
Autoantígenos/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Ribosómicas/biosíntesis , Animales , Autoantígenos/genética , Línea Celular , Humanos , Ratones , Estabilidad del ARN , ARN Mensajero/genética , ARN de Transferencia/genética , Ribonucleoproteínas/genética , Proteínas Ribosómicas/genética , Antígeno SS-BRESUMEN
Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs. Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , Ribonucleasa H/antagonistas & inhibidores , Animales , Secuencia de Bases , ADN/química , ADN/farmacología , Evolución Molecular Dirigida , Inhibidores Enzimáticos/metabolismo , G-Cuádruplex , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Oligonucleótidos Antisentido/farmacología , Biosíntesis de Proteínas , Conejos , Reticulocitos/metabolismo , Ribonucleasa H/metabolismo , Análisis de Secuencia de ADNRESUMEN
La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1(Cre) La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until â¼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Lóbulo Frontal/inmunología , Neuronas/inmunología , Ribonucleoproteínas/inmunología , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Linfocitos B/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/inmunología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Hipocampo/inmunología , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , ARN/genética , ARN/inmunología , ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/inmunología , Precursores del ARN/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/inmunología , ARN de Transferencia/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Síndrome de Sjögren/genética , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Factores de Tiempo , Antígeno SS-BRESUMEN
The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all â¼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.
Asunto(s)
Autoantígenos/química , Autoantígenos/metabolismo , Poli A/metabolismo , Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/metabolismo , Estabilidad del ARN , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al GTP/metabolismo , Técnicas de Silenciamiento del Gen , Biblioteca de Genes , Semivida , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Proteínas de Neoplasias/metabolismo , Péptidos/química , Péptidos/metabolismo , Polirribosomas/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Cinasa C Activada , Receptores de Superficie Celular/metabolismo , Termodinámica , Técnicas del Sistema de Dos Híbridos , Antígeno SS-BRESUMEN
We report here crystal structures of human RNase H1 complexed with an RNA/DNA substrate. Unlike B. halodurans RNase H1, human RNase H1 has a basic protrusion, which forms a DNA-binding channel and together with the conserved phosphate-binding pocket confers specificity for the B form and 2'-deoxy DNA. The RNA strand is recognized by four consecutive 2'-OH groups and cleaved by a two-metal ion mechanism. Although RNase H1 is overall positively charged, the substrate interface is neutral to acidic in character, which likely contributes to the catalytic specificity. Positions of the scissile phosphate and two catalytic metal ions are interdependent and highly coupled. Modeling of HIV reverse transcriptase (RT) with RNA/DNA in its RNase H active site suggests that the substrate cannot simultaneously occupy the polymerase active site and must undergo a conformational change to toggle between the two catalytic centers. The region that accommodates this conformational change offers a target to develop HIV-specific inhibitors.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , VIH/genética , Proteínas de Unión al ARN/química , ADN Polimerasa Dirigida por ARN/química , ARN/química , Transcripción Reversa , Ribonucleasa H/química , Secuencia de Aminoácidos , Calcio/química , Dominio Catalítico , Cristalografía , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH/efectos de los fármacos , VIH/enzimología , VIH/metabolismo , Humanos , Magnesio/química , Manganeso/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Transcripción Reversa/efectos de los fármacos , Ribonucleasa H/metabolismo , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.
Asunto(s)
ADN/química , Iones/química , Metales/química , Ácidos Nucleicos Heterodúplex/química , ARN/química , Ribonucleasa H/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas , Bacillus/química , Bacillus/metabolismo , Secuencia de Bases , Catálisis , Dominio Catalítico/fisiología , Cristalografía por Rayos X , ADN/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , VIH-1/química , VIH-1/metabolismo , Radical Hidroxilo/química , Magnesio/química , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/metabolismo , Hibridación de Ácido Nucleico/fisiología , Estructura Terciaria de Proteína/fisiología , ARN/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasas/química , Ribonucleasas/metabolismo , Especificidad por Sustrato/fisiología , Transposasas/química , Transposasas/metabolismo , Agua/químicaRESUMEN
A capillary electrophoretic assay was developed to measure the ribonuclease (RNase) H activity of human immunodeficiency virus (HIV) type 1 reverse transcriptase. Cleavage of a fluorescein-labeled RNA-DNA heteroduplex was monitored by capillary electrophoresis. This new assay was used as a secondary assay to confirm hits from a high-throughput screening program. Since autofluorescent compounds in samples migrated differently from both substrate and product in most cases, the assay was extremely robust for assaying enzymatic inhibition of such samples, in contrast to a simple well-based approach. The assay was broadly applicable to other RNases H, specifically those from human, Escherichia coli, and HIV-2, although product profiles varied for each enzyme.