Lesley S A, Brow M A, Burgess R R
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
J Biol Chem. 1991 Feb 5;266(4):2632-8.
The interaction of two Escherichia coli transcription factors (sigma 54 and sigma 32) with the core RNA polymerase was studied here. We examined the core binding ability of various fragments of these two transcription factors using a novel method for the in vitro synthesis of truncated proteins in a coupled transcription-translation (S-30) system. The method uses DNA templates generated by the polymerase chain reaction to direct synthesis of precisely truncated fragments of the encoded protein. Primers for the polymerase chain reaction contain transcription and translation signals so that the resulting product can be incubated in the S-30 directly. The synthesized proteins were used for mapping of both functional domains and epitopes of monoclonal antibodies to sigma 54 and sigma 32.
本文研究了两种大肠杆菌转录因子（σ⁵⁴和σ³²）与核心RNA聚合酶的相互作用。我们使用一种在偶联转录-翻译（S-30）系统中体外合成截短蛋白的新方法，检测了这两种转录因子不同片段的核心结合能力。该方法利用聚合酶链反应产生的DNA模板来指导编码蛋白精确截短片段的合成。聚合酶链反应的引物包含转录和翻译信号，以便所得产物可直接在S-30中孵育。合成的蛋白用于绘制针对σ⁵⁴和σ³²的单克隆抗体的功能结构域和表位图谱。
