RESUMEN
Rpp21, a protein subunit of human nuclear ribonuclease P (RNase P) was cloned by virtue of its homology with Rpr2p, an essential subunit of Saccharomyces cerevisiae nuclear RNase P. Rpp21 is encoded by a gene that resides in the class I gene cluster of the major histocompatibility complex, is associated with highly purified RNase P, and binds precursor tRNA. Rpp21 is predominantly localized in the nucleoplasm but is also observed in nucleoli and Cajal bodies when expressed at high levels. Intron retention and splice-site selection in Rpp21 precursor mRNA regulate the intranuclear distribution of the protein products and their association with the RNase P holoenzyme. Our study reveals that dynamic nuclear structures that include nucleoli, the perinucleolar compartment and Cajal bodies are all involved in the production and assembly of human RNase P.
Asunto(s)
Endorribonucleasas/química , ARN Catalítico/química , Células 3T3 , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Western Blotting , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Clonación Molecular , ADN Complementario/metabolismo , Endorribonucleasas/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Intrones , Complejo Mayor de Histocompatibilidad , Ratones , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Empalme del ARN , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleasa P , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
A yeast three-hybrid system was employed to analyze interactions in vivo between H1 RNA, the RNA subunit of human nuclear RNase P, and eight of the protein subunits of the enzyme. The genetic analysis indicates that subunits Rpp21, Rpp29, Rpp30, and Rpp38 interact directly with H1 RNA. The results of direct UV crosslinking studies of the purified RNase P holoenzyme confirm the results of the three-hybrid assay.
Asunto(s)
Núcleo Celular/enzimología , Endorribonucleasas/metabolismo , ARN Catalítico/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Western Blotting , Endorribonucleasas/química , Células HeLa , Humanos , ARN Catalítico/química , Ribonucleasa PRESUMEN
Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.
Asunto(s)
Proteínas Bacterianas/genética , ADN-Topoisomerasas de Tipo II/genética , Endorribonucleasas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos/fisiología , ARN Catalítico/genética , Secuencia de Bases , Girasa de ADN , Escherichia coli/fisiología , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa PAsunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Regulación de la Expresión Génica/efectos de los fármacos , Oligodesoxirribonucleótidos Antisentido/síntesis química , Oligodesoxirribonucleótidos Antisentido/farmacología , Precursores del ARN/metabolismo , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Diseño de Fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Vectores Genéticos , Humanos , Mamíferos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos Antisentido/química , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Precursores del ARN/química , Precursores del ARN/efectos de los fármacos , ARN Bacteriano/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Nuclear Pequeño/genética , ARN de Transferencia de Tirosina/genética , Ribonucleasa P , Especificidad por SustratoRESUMEN
At least six proteins co-purify with human ribonuclease P (RNase P), a tRNA processing ribonucleoprotein. Two of these proteins, Rpp30 and Rpp38, are Th autoantigens. Recombinant Rpp30 and Rpp38 are also recognized by Th sera from systemic sclerosis patients. Two of the other proteins associated with RNase P, Rpp20 and Rpp40, do not cross-react with Th sera. Polyclonal antibodies raised against all four recombinant proteins recognize the corresponding proteins associated with RNase P and precipitate active holoenzyme. Catalytically active RNase P holoenzyme can be separated from the nucleolar and mitochondrial RNA processing endoribonuclease, RNase MRP, even though these two enzymes may share some subunits.
Asunto(s)
Autoantígenos/inmunología , Proteínas Portadoras , Endorribonucleasas/inmunología , ARN Catalítico/inmunología , Ribonucleoproteínas/inmunología , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Autoantígenos/biosíntesis , Autoantígenos/genética , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Endorribonucleasas/biosíntesis , Endorribonucleasas/genética , Células Eucariotas , Humanos , Datos de Secuencia Molecular , Pruebas de Precipitina , ARN Catalítico/biosíntesis , ARN Catalítico/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Ribonucleasa P , Ribonucleoproteínas/biosíntesis , Ribonucleoproteínas/genéticaRESUMEN
Plasmids that contain synthetic genes coding for small oligoribonucleotides called external guide sequences (EGSs) have been introduced into strains of Escherichia coli harboring antibiotic resistance genes. The EGSs direct RNase P to cleave the mRNAs transcribed from these genes thereby converting the phenotype of drug-resistant cells to drug sensitivity. Increasing the EGS-to-target mRNA ratio by changing gene copy number or the number of EGSs complementary to different target sites enhances the efficiency of the conversion process. We demonstrate a general method for the efficient phenotypic conversion of drug-resistant bacterial cultures.
Asunto(s)
Resistencia a Medicamentos/genética , Endorribonucleasas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Fenotipo , ARN Catalítico/genética , Plásmidos/genética , ARN Mensajero/genética , Ribonucleasa P , TransfecciónRESUMEN
Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.
Asunto(s)
Endorribonucleasas/fisiología , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , ARN Catalítico/fisiología , Secuencia de Bases , Escherichia coli/enzimología , Datos de Secuencia Molecular , Oligorribonucleótidos/química , ARN sin Sentido/química , ARN Bacteriano/genética , ARN Mensajero/genética , Ribonucleasa P , TemperaturaRESUMEN
A gel-shift assay was devised to detect stable enzyme-substrate (E-S) complexes between M1 RNA, the catalytic subunit of RNase P from Escherichia coli, and its tRNA precursor substrates. The use of deletion derivatives of M1 RNA in the gel-shift assay has allowed us to identify regions of the enzyme that are involved in the binding of the substrate or that are necessary for catalytic activity. Fragments of substrates that contain the 3' CCA sequence bind preferentially to regions in the 5' half of M1 RNA, while 5' leader sequences interact primarily with regions in the 3' half of M1 RNA. The 5' leader sequence present in the precursor to tRNA(Tyr)su3 from E. coli plays an important role in the formation of stable E-S complexes with M1 RNA. The CCA sequence at the 3' end of precursor tRNA substrates is involved in the product-release step of the reaction that is catalyzed by M1 RNA. Direct measurements of the concentrations of all the components in the reaction catalyzed by M1 RNA facilitated a new approach to the kinetic analysis of the action of the enzyme.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Precursores del ARN/metabolismo , ARN Bacteriano/metabolismo , ARN Catalítico/metabolismo , ARN de Transferencia/metabolismo , Secuencia de Bases , Electroforesis en Gel de Agar , Escherichia coli/metabolismo , Cinética , Metales/farmacología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/efectos de los fármacos , Ribonucleasa P , Especificidad por SustratoRESUMEN
External guide sequences (EGSs) complementary to mRNAs that encode beta-galactosidase from Escherichia coli and nuclease A from Staphylococcus aureus can target these RNAs for cleavage in vitro by RNase P from E. coli. Specific cleavage occurs at locations predicted by the nucleotide sequences of the EGSs. EGSs with regions complementary to the mRNAs that are as short as 13 nucleotides function efficiently and turn over slowly during incubation with the target substrate and the enzyme. EGSs composed of deoxyribonucleotides as well as those composed of ribonucleotides are effective, but cleavage of the targeted substrate with DNA as an EGS is about 10-fold less efficient than that with RNA as an EGS. An RNA EGS inhibited the formation of beta-galactosidase activity in a crude extract (S30) of E. coli that was capable of catalyzing coupled transcription-translation reactions.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , beta-Galactosidasa/genética , Secuencia de Bases , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Cinética , Datos de Secuencia Molecular , Ribonucleasa P , Especificidad por Sustrato , beta-Galactosidasa/metabolismoRESUMEN
Certain fragments of M1 RNA, the catalytic subunit of RNase P from Escherichia coli, either have no enzymatic activity at all or have altered substrate specificity compared with that of the intact catalytic RNA. After simple mixing in vitro, many of these fragments of M1 RNA can reassociate with other fragments to form complexes that have enzymatic activity typical of wild-type M1 RNA. Furthermore, inactive M1 RNA molecules with internal deletions can be complemented in vitro by other inactive derivatives of M1 RNA that have nonoverlapping deletions. Thus, two inactive molecules of M1 RNA can interact to form an active RNA enzyme. Functional attributes can be assigned to various regions of M1 RNA when the reconstitution process is combined with assays for activity with different substrates.
Asunto(s)
Endorribonucleasas/química , Proteínas de Escherichia coli , ARN Catalítico/química , Secuencia de Bases , Catálisis , Fenómenos Químicos , Química Física , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Datos de Secuencia Molecular , Estructura Molecular , ARN Catalítico/metabolismo , Ribonucleasa P , Especificidad por SustratoRESUMEN
In a previous study it was shown that RNase P from E. coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al. (1988) Cell, 53, 267-272). Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain. In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P. Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates. The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al. (1987), J. Biomol. Struct. Dyn. 263, 652-657). Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates. Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , ARN de Transferencia/metabolismo , ARN Viral/metabolismo , Secuencia de Bases , Cinética , Datos de Secuencia Molecular , Virus del Mosaico , Mutación , Conformación de Ácido Nucleico , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN de Transferencia/genética , ARN Viral/genética , Ribonucleasa P , Transcripción GenéticaAsunto(s)
Endorribonucleasas/aislamiento & purificación , Proteínas de Escherichia coli , Escherichia coli/enzimología , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Escherichia coli/genética , Genes Bacterianos , Vectores Genéticos , Indicadores y Reactivos , Sustancias Macromoleculares , Mutación , Plásmidos , Ribonucleasa PRESUMEN
Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other. The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage. The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex. The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA. These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , ARN Bacteriano/genética , Secuencia de Bases , Endorribonucleasas/genética , Escherichia coli/enzimología , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Precursores del ARN/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/genética , Ribonucleasa P , Especificidad por SustratoRESUMEN
RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.
Asunto(s)
Endorribonucleasas/fisiología , Precursores del ARN/metabolismo , Empalme del ARN , ARN/fisiología , Catálisis , Conformación de Ácido Nucleico , ARN Ribosómico 23S/metabolismo , Ribonucleasa P , Especificidad por SustratoRESUMEN
A quasi-continuous double hellix, containing a pseudoknot and ending in a single-stranded region which contains CCA, can be formed at the 3' terminus of the genomic RNAs of certain plant viruses. M1 RNA (the catalytic subunit) alone and the RNAase P holoenzyme from E. coli cleave the tRNA-like structure of TYMV RNA in vitro at the 5' side of the quasi-helical structure to generate 5' phosphate and 3' hydroxyl groups in the cleavage products. The intact pseudoknot structure in the substrate is not required for the reaction catalyzed by M1 RNA alone, but its presence markedly improves the efficiency of the reaction. In addition to its essential role in the biosynthesis of tRNA, RNAase P may have another function in vivo, namely, in the physiology of viral infections.
Asunto(s)
Endorribonucleasas/metabolismo , Virus del Mosaico/genética , ARN de Transferencia/metabolismo , ARN Viral/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ribonucleasa P , Especificidad por SustratoRESUMEN
M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Precursores del ARN/metabolismo , Secuencia de Bases , ADN/genética , ADN Recombinante , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Plásmidos , ARN Bacteriano/genética , Aminoacil-ARN de Transferencia/genética , Ribonucleasa P , Ribonucleasa T1/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Supresión GenéticaRESUMEN
We have isolated a weak UGA suppressor of phage T4 tRNA(Gly) in which the anticodon is changed from UCC to UCA. Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA(Gly). Both mutations change the T stem of the cloverleaf model. One involved a G to A change at the 5' base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop. The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P. Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.
Asunto(s)
Mutación , Aminoacil-ARN de Transferencia/genética , ARN Viral/genética , Supresión Genética , Fagos T/genética , Secuencia de Bases , Precursores de Ácido Nucleico/metabolismo , ARN/metabolismo , Precursores del ARNAsunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Halobacterium/enzimología , ARN Bacteriano/metabolismo , Animales , Bovinos , Núcleo Celular/enzimología , Endorribonucleasas/genética , Endorribonucleasas/aislamiento & purificación , Genes , Genes Bacterianos , Células HeLa/enzimología , Humanos , Miocardio/enzimología , Conformación de Ácido Nucleico , Ribonucleasa P , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Truncated transcripts of the rnpB gene from E. coli, coding for M1 RNA, the catalytic subunit of RNAase P, and fragments of M1 RNA generated by nuclease treatment have been prepared, and their ability to function catalytically in vitro has been determined. Molecules missing as many as 122 nucleotides at the 3' terminus retain catalytic activity, although at a much lower level than M1 RNA itself. No activity is observed with an RNA that is missing 70 nucleotides at the 5' terminus. The removal of even a small number of nucleotides from both termini eliminates all catalytic function. The preservation of one intact terminus may be essential for the tertiary and quaternary interactions required to generate the conformation of an active RNA species.
Asunto(s)
Proteínas Bacterianas/genética , Endorribonucleasas/genética , ARN Bacteriano/genética , ARN Mensajero/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Deleción Cromosómica , Endorribonucleasas/metabolismo , Activación Enzimática , Cinética , Conformación de Ácido Nucleico , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa P , Ribonucleasas , Transcripción GenéticaRESUMEN
M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P. M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate. Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA. Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions. The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol. Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.