Gaillard C, Strauss F
Institut Jacques Monod, Paris, France.
J Mol Biol. 1990 Sep 20;215(2):245-55. doi: 10.1016/S0022-2836(05)80343-2.
We have detected, in nuclear extracts of non-infected cultured monkey cells, a protein (protein H16) that binds a specific single-stranded DNA sequence in the early promoter of simian virus 40 (SV40). This protein does not bind double-stranded DNA, nor RNA. In the present paper, the DNA-binding properties of protein H16 and its effects on transcription by RNA polymerase II in vitro have been investigated. The protein binds only to the late strand of the early promoter, within the region of the 21 base-pair repeats, and shows no affinity for any other SV40 sequence. The high percentage of cytosine residues in the late strand in this region appears to be important for recognition by the protein. Protein H16 does not bind the control region of SV40 in negatively supercoiled DNA circles. When bound to the late strand, the protein is displaced from its binding site by reassociation of the early strand with the late strand. Its binding to DNA is not sensitive to methylation of the dinucleotide CG in its binding site. The protein has been purified to near homogeneity by preparative gel retardation, and has an apparent molecular weight of 70,000. Purified protein H16 stimulates transcription by purified RNA polymerase II in vitro. The possible role of sequence-specific single-strand-binding proteins in transcription is discussed.
我们在未感染的培养猴细胞核提取物中检测到一种蛋白质（蛋白质H16），它能结合猴病毒40（SV40）早期启动子中的特定单链DNA序列。这种蛋白质不结合双链DNA，也不结合RNA。在本文中，我们研究了蛋白质H16的DNA结合特性及其对体外RNA聚合酶II转录的影响。该蛋白质仅结合早期启动子的晚期链，位于21个碱基对重复序列区域内，对任何其他SV40序列均无亲和力。该区域晚期链中胞嘧啶残基的高比例似乎对蛋白质的识别很重要。蛋白质H16不结合负超螺旋DNA环中的SV40控制区域。当与晚期链结合时，早期链与晚期链重新结合会使该蛋白质从其结合位点上被置换下来。其与DNA的结合对其结合位点中二核苷酸CG的甲基化不敏感。通过制备性凝胶阻滞法已将该蛋白质纯化至接近均一，其表观分子量为70,000。纯化的蛋白质H16在体外刺激纯化的RNA聚合酶II的转录。本文还讨论了序列特异性单链结合蛋白在转录中的可能作用。
