突变型T7 RNA聚合酶能够催化DNA引物延伸反应。

Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction.

作者信息

Rusakova E E, Tunitskaya V L, Memelova L V, Kochetkova S V, Kostyuk D A, Kochetkov S N

机构信息

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.

出版信息

FEBS Lett. 1998 Feb 20;423(2):189-92. doi: 10.1016/s0014-5793(98)00058-1.

DOI:10.1016/s0014-5793(98)00058-1

PMID:9512355

Abstract

The mutant T7 RNA polymerase (T7 RNAP), containing two substitutions (Y639F, S641A) was earlier shown to utilize both rNTP and dNTP in a transcription-like reaction. In this report the ability of the enzyme to catalyze DNA primer extension reaction was demonstrated. The efficiency of the reaction essentially depended on the type of the primer sequence, and was significantly higher if the primer coincided with the T7 promoter non-coding sequence. In this case the primer extension reaction proceeded along with de novo RNA synthesis. The length of the product did not exceed 8 nucleotides, indicating that the primer extension reaction proceeds according to the mechanism of the T7 RNAP-catalyzed abortive transcription.

摘要

含有两个替换位点（Y639F、S641A）的突变型T7 RNA聚合酶（T7 RNAP）此前已被证明在类似转录的反应中能同时利用核糖核苷三磷酸（rNTP）和脱氧核糖核苷三磷酸（dNTP）。在本报告中，该酶催化DNA引物延伸反应的能力得到了证实。反应效率主要取决于引物序列的类型，如果引物与T7启动子非编码序列一致，则效率显著更高。在这种情况下，引物延伸反应与从头RNA合成同时进行。产物长度不超过8个核苷酸，表明引物延伸反应是按照T7 RNAP催化的流产转录机制进行的。