Schmidt R P, Kreuzer K N
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1992 Jun 5;267(16):11399-407.
The bacteriophage T4-encoded MotA protein is critical for transcription from T4 middle-mode promoters. However, a direct interaction of this protein with a middle-mode promoter has not previously been demonstrated. We have cloned the motA gene and overexpressed the gene product using the T7 expression system. A simple procedure was then developed to purify the MotA protein to homogeneity. Using the purified protein we have demonstrated that MotA protein binds to the -30 region of the middle-mode promoter PuvsY. This promoter has previously been shown to be a necessary component of a T4 replication origin, and thus MotA is also a T4 origin-binding protein. Modified RNA polymerase purified from T4-infected cells was used to establish middle-mode transcription in vitro. Transcription from PuvsY was markedly enhanced by the addition of MotA protein, whether or not the template contained the cytosine modifications characteristic of T4 DNA. However, transcription from PuvsY was apparently independent of the MotA protein when unmodified RNA polymerase from uninfected cells was used.
噬菌体T4编码的MotA蛋白对于T4中模式启动子的转录至关重要。然而，此前尚未证明该蛋白与中模式启动子存在直接相互作用。我们克隆了motA基因，并使用T7表达系统对基因产物进行了过表达。随后开发了一种简单的方法将MotA蛋白纯化至同质。利用纯化后的蛋白，我们证明了MotA蛋白与中模式启动子PuvsY的 -30区域结合。该启动子此前已被证明是T4复制起点的必要组成部分，因此MotA也是一种T4起点结合蛋白。从T4感染细胞中纯化得到的经修饰的RNA聚合酶用于在体外建立中模式转录。无论模板是否含有T4 DNA特有的胞嘧啶修饰，添加MotA蛋白均可显著增强PuvsY的转录。然而，当使用未感染细胞的未修饰RNA聚合酶时，PuvsY的转录显然不依赖于MotA蛋白。
