Botfield M C, Weiss M A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1994 Mar 8;33(9):2349-55. doi: 10.1021/bi00175a001.
The POU motif defines a family of eukaryotic transcription factors broadly involved in tissue-specific gene expression and developmental regulation. The motif contains two DNA-binding domains: an N-terminal POU-specific domain (POUs) and C-terminal homeodomain (POUHD). Surprisingly, POUs has recently been found to be similar in structure to helix-turn-helix (HTH) domains of phage repressor and Cro proteins [Assa-Munt, N., Mortishire-Smith, R., Aurora, R., Herr, W., & Wright, P.E. (1993) Cell 73, 193-205; Dekker, N., Cox, M., Boelens, R., Verrijzer, C.P., van der Vliet, P.C., & Kaptein, R. (1993) Nature 362, 852-855]. Because POUHD and POUs are expected to bind DNA differently, we have used "methylphosphonate interference" to investigate the alignment of their HTH elements in a specific DNA complex. This neutral phosphate analogue, originally developed for applications in antisense drug design [Miller, P. S., & Ts'o, P. O. P. (1987) Anti-Cancer Drug Des. 2, 117-128], is shown to provide a sensitive probe for sites of backbone-specific protein-DNA interaction. Inferred POUs-phosphate contacts are in striking accord with cocrystal structures of bacteriophage repressor and Cro proteins. Alignment of POUHD and POUs in successive major grooves in each case predicts unique HTH-adenine contacts. This prediction is verified using DNA base analogues to effect interchange of AT functional groups by the method of 2'-deoxyinosine/5-methyl-2'-deoxycytosine substitution [McLaughlin, L. W., Benseler, F., Graeser, E., Piel, N., & Scholtissek, S. (1987) Biochemistry 26, 7238-7245]. Our results strongly support the hypothesis that the DNA-binding properties of POUs are analogous to those of bacteriophage lambda repressor.
POU基序定义了一类真核转录因子家族，广泛参与组织特异性基因表达和发育调控。该基序包含两个DNA结合结构域：一个N端POU特异性结构域（POUs）和C端同源结构域（POUHD）。令人惊讶的是，最近发现POUs在结构上与噬菌体阻遏蛋白和Cro蛋白的螺旋-转角-螺旋（HTH）结构域相似[阿萨-蒙特，N.，莫蒂希尔-史密斯，R.，奥罗拉，R.，赫尔，W.，& 赖特，P.E.（1993年）《细胞》73卷，193 - 205页；德克尔，N.，考克斯，M.，博伦斯，R.，韦里泽尔，C.P.，范德弗利特，P.C.，& 卡普泰因，R.（1993年）《自然》362卷，852 - 855页]。由于预计POUHD和POUs与DNA的结合方式不同，我们使用“甲基膦酸酯干扰”来研究它们的HTH元件在特定DNA复合物中的排列。这种中性磷酸类似物最初是为反义药物设计而开发的[米勒，P.S.，& 曹，P.O.P.（1987年）《抗癌药物设计》2卷，117 - 128页]，已被证明可为骨架特异性蛋白质 - DNA相互作用位点提供灵敏的探针。推断出的POUs - 磷酸接触与噬菌体阻遏蛋白和Cro蛋白的共晶体结构惊人地一致。在每种情况下，POUHD和POUs在连续的大沟中的排列预测了独特的HTH - 腺嘌呤接触。使用DNA碱基类似物通过2'-脱氧肌苷/5 - 甲基 - 2'-脱氧胞嘧啶取代法[麦克劳克林，L.W.，本塞勒，F.，格雷泽，E.，皮尔，N.，& 朔尔蒂塞克，S.（1987年）《生物化学》26卷，7238 - 7245页]实现AT功能基团的互换，验证了这一预测。我们的结果有力地支持了POUs的DNA结合特性与噬菌体λ阻遏蛋白的DNA结合特性相似的假说。
