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Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
actividad de la helicasa de ARNm del ribosoma
Seyedtaghi Takyar1, Robyn P Hickerson, Harry F Noller
1Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.
Cell
|January 18, 2005
Resumen
El ribosoma actúa como una helicasa de ARNm, desenrolando las estructuras secundarias que impiden la traducción. Esta actividad recién identificada, localizada en la subunidad 30S, involucra a las proteínas S3 y S4.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- La bioquímica es la bioquímica.
- Genética La genética.
Sus antecedentes:
- Las estructuras secundarias del ARN mensajero (ARNm) representan una barrera a la traducción.
- Los ribosomas deben navegar por estas estructuras para la síntesis de proteínas.
- Investigaciones anteriores no habían identificado una actividad específica de helicasa responsable de este proceso.
Objetivo del estudio:
- Investigar el mecanismo por el cual los ribosomas se traducen a través de estructuras secundarias de ARNm.
- Para identificar cualquier actividad de helicasa asociada con el ribosoma.
- Para localizar el sitio activo e identificar los componentes proteicos clave involucrados.
Principales métodos:
- Análisis de desplazamiento de oligonucleótidos.
- Paso a paso en el sistema de traducción in vitro con componentes purificados.
- Análisis de las interacciones ribosoma-hélice utilizando diferentes longitudes de hélice y registros.
- Mutagénesis dirigida al sitio de las proteínas ribosómicas S3 y S4.
Principales resultados:
- Los ribosomas exhiben actividad de helicasa, interrumpiendo las estructuras secundarias de ARNm aguas abajo, incluida una hélice de 27 pares de bases.
- El sitio activo de la helicasa está localizado en el centro del túnel aguas abajo en la subunidad ribosómica 30S.
- Las mutaciones en las proteínas ribosómicas S3 y S4 afectan significativamente esta actividad de la helicasa.
Conclusiones:
- El ribosoma posee una actividad intrínseca de helicasa de ARNm esencial para la traducción.
- Las proteínas ribosómicas S3 y S4 están implicadas en la función y la procesividad de la mRNA helicasa del ribosoma.
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