Sun W, Hattman S, Kool E
Department of Biology, University of Rochester, NY 14627, USA.
J Mol Biol. 1997 Nov 7;273(4):765-74. doi: 10.1006/jmbi.1997.1349.
The bacteriophage Mu C gene encodes a 16.5 kDa site-specific DNA binding protein that is a transcriptional activator of the four "late" promoters, Pmom, Plys, PI and PP. A symmetrical consensus C recognition sequence, TTAT[N5-6]ATAA, containing an inverted tetrad repeat separated by a spacer of five to six G+C-rich nucleotides, has been proposed. To investigate this, we used oligonucleotide mutagenesis to introduce random substitutions within and flanking the proposed C-target region; each variant site was tested for C recognition by an in vivo functional transactivation assay. We observed that all single mutations, in either tetrad, reduced C activation. Although two out of ten substitutions within the spacer reduced activation, the spacer region does not appear to make specific contact with C. We also used in vitro chemical-protection and -interference to study C contacts with Pmom. The results indicate that C contacts Pmom DNA on only one face of the helix through interactions within two adjacent major grooves; this conclusion was supported by gel shift analyses using synthetic oligonucleotide duplexes containing I.C or other base-pair substitutions. Evidence is also presented that C-Pmom contacts are asymmetrical, and that they extend two nucleotides 3' to the promoter-proximal tetrad. We also show that C binding induces a deformation, possibly a bend, in Pmom DNA.
噬菌体Mu C基因编码一种16.5 kDa的位点特异性DNA结合蛋白，它是四个“晚期”启动子Pmom、Plys、PI和PP的转录激活因子。有人提出了一个对称的共有C识别序列TTAT[N5 - 6]ATAA，其中包含一个由五到六个富含G + C的核苷酸间隔开的反向四重重复序列。为了对此进行研究，我们使用寡核苷酸诱变在提议的C靶区域内及其侧翼引入随机取代；通过体内功能反式激活测定法测试每个变异位点的C识别情况。我们观察到，在任一四重重复序列中的所有单突变都会降低C的激活作用。虽然间隔区内十个取代中有两个降低了激活作用，但间隔区似乎并未与C进行特异性接触。我们还使用体外化学保护和干扰来研究C与Pmom的接触。结果表明，C仅通过两个相邻大沟内的相互作用在螺旋的一个面上与Pmom DNA接触；使用含有I.C或其他碱基对取代的合成寡核苷酸双链体进行的凝胶迁移分析支持了这一结论。还有证据表明，C与Pmom的接触是不对称的，并且它们在启动子近端四重重复序列的3'端延伸两个核苷酸。我们还表明，C的结合会诱导Pmom DNA发生变形，可能是弯曲。
