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Eventos resueltos en el tiempo en la vía de reacción de iniciación de transcripción por una polimerasa de ARN de una

Yuanyuan Chen¹, Ritwika Basu, Michael L Gleghorn

¹Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, United States.

Journal of the American Chemical Society

|July 13, 2011

Resumen

Este estudio utilizó la microscopia Raman para observar la reacción de transferencia de nucleotidilo en tiempo real en la ARN polimerasa (ARNP) del fago N4. Los hallazgos revelan pasos clave en la formación de enlaces fosfodiéster y cambios en la conformación de las enzimas durante la catálisis.

Área de la Ciencia:

La bioquímica es la bioquímica.
Biología Molecular Biología Molecular
Biología Estructural Biología estructural.

Sus antecedentes:

La ARN polimerasa del fago N4 (RNAP) es crucial para la replicación del ARN viral.
Comprender la cinética de la formación de enlaces fosfodiéster es clave para comprender la función de la RNAP.

Objetivo del estudio:

Para monitorear en tiempo real la reacción de transferencia de nucleotidilo en cristales simples de RNAP del fago N4.
Para dilucidar los cambios conformacionales y la dinámica de unión del sustrato/metal durante la catálisis.

Principales métodos:

Microscopía Raman en tiempo real de cristales complejos RNAP-ADN individuales.
En cristallo empapado de sustratos de nucleósido trifosfato (NTP) y cationes divalentes.
Utilizando NTP etiquetados isotópicamente y datos de cristalografía de rayos X para la asignación espectral.

Principales resultados:

Se observa la unión al sustrato en 2-7 minutos y cambios de conformación de la hélice O.
Cambios en la conformación de la unión de metal divalente y el trifosfato de ribosa.
La formación de enlaces de fosfodiéster se completa en ~ 15 min, con la liberación de metal en ~ 40 min.

Conclusiones:

El movimiento de la hélice O puede ser inducido únicamente por la unión del sustrato.
La cinética de la reacción in crystallo revela intermediarios transitorios para un estudio más detallado.
Este trabajo proporciona una visión dinámica de la catálisis de RNAP a nivel molecular.

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