RESUMEN
Tyrosinases from various organisms are compared with respect to enzymatic structure, primary, secondary and tertiary structure, domain structure, Cu binding sites, maturation mechanism and activation mechanism. On the basis of these comparisons, and by using hemocyanin structure as a template, a structure model for the active site of tyrosinases is proposed.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hongos/enzimología , Monofenol Monooxigenasa/química , Monofenol Monooxigenasa/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/enzimología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The structure of the conserved region of the U1A pre-mRNA (AgRNA) and its complex with U1A protein was investigated. The previously proposed secondary structure of Ag RNA, derived from enzymatic probing and analysis of the structure and function of mutant mRNAs, is now confirmed by chemical probing data and further refined in the regions where the enzymatic data were not conclusive. The two unpaired nucleotides in the internal loops opposite of the Box sequences as well as the tetraloop could not be cleaved by ribonucleases, but are accessible to chemical probes. Concerning the RNA-protein complex, the protection experiments showed that the Box regions are largely protected when the U1A protein is present. All stem regions in the 5' part of the structure seem protected against ribonucleases. Unexpectedly, the nucleotides of the tetraloop become accessible to ribonucleases in the RNA-protein complex. This result indicates that the tetraloop undergoes a conformational change upon U1A protein binding. The 3' part of the Ag RNA sequence, containing the polyadenylation signal in a hairpin structure, showed hardly any protection, a finding that agrees with the fact that U1A does not interfere with the binding of the cleavage polyadenylation specificity factor (CPSF) to the polyadenylation signal.
Asunto(s)
Huella de ADN , Conformación de Ácido Nucleico , ARN Mensajero/química , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U1/genética , Composición de Base , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Precursores del ARN/química , Proteínas de Unión al ARN/química , Ribonucleoproteína Nuclear Pequeña U1/química , Ribonucleoproteína Nuclear Pequeña U1/aislamiento & purificaciónRESUMEN
The secondary structures of human hY1 and hY5 RNAs were determined using both chemical modification techniques and enzymatic structure probing. The results indicate that both for hY1 and for hY5 RNA the secondary structure largely corresponds to the structure predicted by sequence alignment and computerized energy-minimization. However, some important deviations were observed. In the case of hY1 RNA, two regions forming a predicted helix appeared to be single-stranded. Furthermore, the pyrimidine-rich region of hY1 RNA appeared to be very resistant to reagents under native conditions, although it was accessible to chemical reagents under semi-denaturing conditions. This may point to yet unidentified tertiary interactions for this region of hY1 RNA. In the case of hY5 RNA, two neighbouring internal loops in the predicted structure appeared to form one large internal loop.
Asunto(s)
Autoantígenos/genética , Conformación de Ácido Nucleico , ARN Ribosómico/química , ARN Citoplasmático Pequeño , Ribonucleoproteínas/genética , Animales , Autoantígenos/metabolismo , Secuencia de Bases , Clonación Molecular , ADN , Humanos , Datos de Secuencia Molecular , Sondas ARN , ARN Ribosómico/metabolismo , Ribonucleoproteínas/metabolismo , Alineación de SecuenciaAsunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Núcleo Celular/inmunología , Epítopos/inmunología , Enfermedades Reumáticas/inmunología , Anticuerpos Antinucleares/inmunología , Secuencia de Bases , Mapeo Cromosómico , Epítopos/genética , Humanos , Datos de Secuencia Molecular , Enfermedades Reumáticas/genética , Linfocitos T/inmunologíaRESUMEN
Generation of full protein coordinates from limited information, e.g., the C alpha coordinates, is an important step in protein homology modeling and structure determination, and molecular dynamics (MD) simulations may prove to be important in this task. We describe a new method, in which the protein backbone is built quickly in a rather crude way and then refined by minimization techniques. Subsequently, the side chains are positioned using extensive MD calculations. The method is tested on two proteins, and results compared to proteins constructed using two other MD-based methods. In the first method, we supplemented an existing backbone building method with a new procedure to add side chains. The second one largely consists of available methodology. The constructed proteins are compared to the corresponding X-ray structures, which became available during this study, and they are in good agreement (backbone RMS values of 0.5-0.7 A, and all-atom RMS values of 1.5-1.9 A). This comparative study indicates that extensive MD simulations are able, to some extent, to generate details of the native protein structure, and may contribute to the development of a standardized methodology to predict reliably (parts of) protein structures when only partial coordinate data are available.
Asunto(s)
Proteínas/química , Proteínas de Unión al ARN , Aminoácidos/química , Simulación por Computador , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Fosfopiruvato Hidratasa/química , Conformación Proteica , Ribonucleoproteína Nuclear Pequeña U1/química , TermodinámicaRESUMEN
The human U1A protein-U1A pre-mRNA complex and the relationship between its structure and function in inhibition of polyadenylation in vitro were investigated. Two molecules of U1A protein were shown to bind to a conserved region in the 3' untranslated region of U1A pre-mRNA. The secondary structure of this region was determined by a combination of theoretical prediction, phylogenetic sequence alignment, enzymatic structure probing and molecular genetics. The U1A binding sites form (part of) a complex secondary structure which is significantly different from the binding site of U1A protein on U1 snRNA. Studies with mutant pre-mRNAs showed that the integrity of much of this structure is required for both high affinity binding to U1A protein and specific inhibition of polyadenylation in vitro. In particular, binding of a single molecule of U1A protein to U1A pre-mRNA is not sufficient to produce efficient inhibition of polyadenylation.