Análisis y modelado estructural de los dominios catalíticos de DCL1 de Arabidopsis thaliana
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Los micro-ARNs (miARN) son una clase de ARNs pequeños que participan
activamente de procesos biológicos fundamentales tales como el desarrollo de los
organismos y las respuestas a cambios ambientales. Su biogénesis es un proceso
complejo que comienza con el procesamiento de precursores que no poseen
secuencias en común. La información estructural de alta resolución sobre proteínas
involucradas en el proceso es escasa. La endoribonucleasa DCL1 es la enzima más
importante involucrada en el proceso en plantas; sin embargo, no se conoce en
detalle su funcionamiento. El estudio estructural y funcional de proteínas
multidominio como DCL1 requiere la expresión por separado de cada dominio.
Conocer el plegamiento de la proteína en estudio permitiría realizar elecciones
racionales en la definición de los límites de los constructos a expresar. Por todo esto
se planteó en este trabajo hacer un análisis estructural in silico de la región
Plataforma-PAZ de DCL1, que es esencial en la determinación del tamaño del
producto.
Para ello se comenzó generando una base de datos con secuencias de
proteínas que tuvieran similitud con la homóloga humana DICER, la cual fue curada
según parámetros de similitud. Luego se procedió a realizar una predicción de
dominios, en base a los cuales se determinó una región de interés que fue extraída
de cada una de las secuencias. Después de realizar un análisis de la identidad
presente entre las mismas, se extrajo un subgrupo de secuencias el cual fue
alineado. Se llevó a cabo la predicción de estructuras secundarias de algunas de las
proteínas de interés y se comparó con la información presente en bibliografía. Se
pudo observar que las predicciones tanto de dominios como de secuencias fueron
muy buenas, correspondiéndose con lo esperado. Sin embargo el alineamiento no
fue bueno, y esto puede deberse a la baja homología de secuencia presente entre
las distintas proteínas. Finalmente se hizo un modelado estructural usando la
información del alineamiento general y la estructura conocida de la homóloga
humana.
Micro-RNAs (miRNAs) are a class of small RNAs that actively participate in fundamental biological processes such as the development of organisms and responses to environmental changes. Its biogenesis is a complex process that begins with the processing of precursors that do not have sequences in common. The high resolution structural information on proteins involved in the process is scarce. The endoribonuclease DCL1 is the most important enzyme involved in miRNA processing in plants; however, there is little experimental information on the enzyme structure and activity. Structural and functional studies of multidomain proteins such as DCL1 require the expression of isolated domain(s). Knowledge of the folding of the protein under study allows rational choices in the definition of the limits of the constructs to be expressed. In this work we propose to perform an insilico structural analysis of the DCL1 Platform-PAZ region, essential in the determination of the small RNA product size. With this purpose, a database was created with protein sequences that show similarity to the human homologue DICER. The database was cured according to similarity parameters. A prediction of domain architecture was performed, based on which a region of interest was determined. This region was extracted from each of the sequences. After performing an analysis of the identity between them, a subgroup of sequences was extracted and aligned. Prediction of secondary structures of some of the proteins of interest was carried out, and then compared with the information present in the literature. It was observed that the predictions of domains and sequences were very good, according to what was expected. However, the alignment was not good, probably due to the low sequence homology present between the different proteins. Finally, a structural model of the region of interest was obtained using the information of the general alignment and the known structure of the human homologue.
Micro-RNAs (miRNAs) are a class of small RNAs that actively participate in fundamental biological processes such as the development of organisms and responses to environmental changes. Its biogenesis is a complex process that begins with the processing of precursors that do not have sequences in common. The high resolution structural information on proteins involved in the process is scarce. The endoribonuclease DCL1 is the most important enzyme involved in miRNA processing in plants; however, there is little experimental information on the enzyme structure and activity. Structural and functional studies of multidomain proteins such as DCL1 require the expression of isolated domain(s). Knowledge of the folding of the protein under study allows rational choices in the definition of the limits of the constructs to be expressed. In this work we propose to perform an insilico structural analysis of the DCL1 Platform-PAZ region, essential in the determination of the small RNA product size. With this purpose, a database was created with protein sequences that show similarity to the human homologue DICER. The database was cured according to similarity parameters. A prediction of domain architecture was performed, based on which a region of interest was determined. This region was extracted from each of the sequences. After performing an analysis of the identity between them, a subgroup of sequences was extracted and aligned. Prediction of secondary structures of some of the proteins of interest was carried out, and then compared with the information present in the literature. It was observed that the predictions of domains and sequences were very good, according to what was expected. However, the alignment was not good, probably due to the low sequence homology present between the different proteins. Finally, a structural model of the region of interest was obtained using the information of the general alignment and the known structure of the human homologue.
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