RESUMEN
The tRNA splicing endonuclease cleaves intron-containing tRNA precursors on both sides of the intron. The prevailing belief has been that the enzyme binds only to the mature domain through the invariant bases. We show instead that, for recognition, the endonuclease utilizes distinct sets of structural elements, several of which are within the intron. One subset of recognition elements, localized in the mature domain, is needed for recognition of both cleavage sites, while two other subsets, localized at the exon-intron boundaries, are used for recognition of either one or the other cleavage site. The two cleavage sites are essentially independent: neither is required by the other for cleavage to take place. These results support a two-active-site model for the eucaryal endonuclease.
Asunto(s)
Endorribonucleasas/metabolismo , Precursores del ARN/química , Precursores del ARN/metabolismo , Levaduras/genética , Secuencia de Bases , Células Eucariotas/fisiología , Exones/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Intrones/fisiología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Empalme del ARN/fisiología , Especificidad por Sustrato , Levaduras/enzimologíaRESUMEN
A single tRNA-splicing endoribonuclease can cleave several precursors. In addition to the conserved nucleotides (nt), there are positions in the mature domain that, though not always occupied by the same nt, nevertheless play a fundamental role in intron excision reaction. The elements of the recognition set (invariant nt, nt at the cardinal positions) can contribute to the overall recognition process by providing either a direct contact for the enzyme or by dictating the spatial orientation of nt that are directly contacted.
Asunto(s)
Endorribonucleasas/metabolismo , Empalme del ARN , ARN de Transferencia/metabolismo , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia/químicaAsunto(s)
Endorribonucleasas/aislamiento & purificación , Oocitos/enzimología , Animales , Núcleo Celular/enzimología , Centrifugación por Gradiente de Densidad/métodos , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular , Detergentes , Endorribonucleasas/metabolismo , Femenino , Indicadores y Reactivos , Cinética , Octoxinol , Radioisótopos de Fósforo , Plásmidos , Polietilenglicoles , Pronasa , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Leucina/metabolismo , Técnica de Dilución de Radioisótopos , Xenopus laevisRESUMEN
To investigate the mechanism by which the purified Xenopus tRNA splicing endonuclease recognizes its splice sites, we utilized yeast pre-tRNA(3Leu) and pre-tRNA(Phe) variants constructed by in vitro mutagenesis. We found that the endonuclease interacts with conserved features of the mature tRNA domain. In particular, U8 and C56 may be examples of contact points between protein and RNA. Given that there are no conserved sequences at the splice junctions, the specificity of cutting at both splice sites is determined by the length of the anticodon stem. Although in general, the sequence of the intron is unimportant for splicing, there are some structural requirements.
Asunto(s)
Endorribonucleasas/metabolismo , Xenopus laevis/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Intrones , Conformación de Ácido Nucleico , Plásmidos , Precursores del ARN/metabolismo , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Xenopus laevis/genéticaRESUMEN
A simple technique is described that allows mixed populations of eukaryotic cells to be screened for clones containing multiple copies of a particular DNA. Essentially, eukaryotic cells are transferred to either nitrocellulose of Whatman 541 filters, and their DNA is immobilised in situ. Exposure of the filters to a 32P-labeled DNA "probe" results in detectable hybridisation only at the positions of clones containing multiple copies of the DNA. Using Whatman 541 paper, a portion of the cells, evenly distributed throughout the mixed population is retained on the culture dish, and can be propagated further for subsequent cell cloning. The technique has allowed rapid distinction of clones of transformed rat cells that contain a single or only a few copies per cell of polyoma viral DNA from clones maintaining multiple copies. The technique has also been used to distinguish between clones of mouse L-cells containing multiple and only a few copies of 0X174 DNA. In this manner the technique allows rapid detection of cells amplifying a particular species of DNA. Finally, the method can be used to detect cells assimilating many copies of a foreign DNA, even in the absence of a co-transfected selectable marker.
Asunto(s)
Autorradiografía/métodos , Replicación del ADN , ADN Recombinante/análisis , Animales , Bacteriófago phi X 174/genética , Secuencia de Bases , Transformación Celular Viral , Células Clonales/metabolismo , ADN/análisis , ADN Viral/análisis , Células L/metabolismo , RatonesAsunto(s)
Transformación Celular Viral , ADN Viral/genética , Genes Virales , Poliomavirus/genética , Animales , Antígenos de Neoplasias/genética , Antígenos Virales/genética , Regulación de la Expresión Génica , Ratones , Peso Molecular , ARN Mensajero/genética , ARN Viral/genética , Ratas , Recombinación Genética , Proteínas Virales/genéticaRESUMEN
A soluble cell-free extract derived from stage 6 Xenopus laevis oocytes is described. From supercoiled simian virus 40 DNA the extract produces nicked circles (having a single-strand scission), linear molecules of full unit size, shorter length fragments, and various forms of complex DNA.