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High-throughput Purification of Affinity-tagged Recombinant Proteins
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Distinguir entre los mecanismos de activación transcripcional de los eucariotas con la ARN polimerasa T7 de los
Cell
|September 25, 1987
Resumen
Los elementos promotores de la levadura se probaron con la polimerasa de ARN T7 de los bacteriófagos para comprender la activación transcripcional. El elemento ded1 activó la transcripción, lo que sugiere efectos de la cromatina, mientras que el elemento gal no lo hizo, lo que indica interacciones específicas de proteínas.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- Genética La genética.
- La bioquímica es la bioquímica.
Sus antecedentes:
- Los mecanismos de activación transcripcional de los eucariotas son complejos y no se comprenden completamente.
- Investigar los elementos promotores con sistemas heterólogos puede dilucidar distintas vías de activación.
Objetivo del estudio:
- Para diferenciar entre los mecanismos de activación de la transcripción eucariota.
- Para determinar si los elementos promotores aguas arriba de la levadura pueden estimular la transcripción mediada por la polimerasa de ARN T7 de los bacteriófagos.
Principales métodos:
- Colocando los elementos promotores aguas arriba gal y ded1 de la levadura aguas arriba de un promotor T7 y su gen 3 en la levadura.
- La producción de polimerasa de ARN T7 de los bacteriófagos dentro de las células de levadura para impulsar la transcripción.
- Cuantificación de los niveles de transcripción de genes his3 bajo diferentes condiciones de elementos promotores.
Principales resultados:
- El elemento gal no estimuló la transcripción his3 por la ARN polimerasa T7, independientemente de su estado de activación normal.
- El elemento ded1 poly ((dA-dT) estimuló significativamente su transcripción aproximadamente 7 veces.
- Esta estimulación por el elemento ded1 fue comparable a su efecto sobre el promotor ded1 nativo.
Conclusiones:
- La activación por el elemento ded1 probablemente implica facilitar la entrada de la maquinaria de transcripción a través de los efectos de la plantilla de cromatina.
- La activación por el elemento gal puede depender de interacciones específicas entre la proteína GAL4 y la maquinaria de transcripción, que la ARN polimerasa T7 no soporta.
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