Chen S, Supakar P C, Vellanoweth R L, Song C S, Chatterjee B, Roy A K
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio 78284-7762, USA.
Mol Endocrinol. 1997 Jan;11(1):3-15. doi: 10.1210/mend.11.1.9868.
The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (approximately 90-bp) homopurine/homopyrimidine (pur/ pyr) stretch immediately upstream of the Sp1-binding GC box site. This pur-pyr stretch is conserved at the same proximal position in the rat, mouse, and human AR gene promoters. Mutation of this region results in a 3-fold decline in promoter activity, indicating an important regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease showed that it is capable of forming a non-B DNA structure involving unpaired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analyses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double-strand DNA-binding protein. Both oligonucleotide cross-competition and antibody supershift experiments established that the double-strand binding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) footprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. These results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant stabilization of the non-B DNA structure is expected to prevent its interaction with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Sp1 sites at the pur/pyr region adjacent to the GC box can provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initiation complex.
雄激素受体（AR）基因启动子不含TATA盒或CAAT盒，但在紧邻Sp1结合GC盒位点的上游含有一段长约90个碱基对的同嘌呤/同嘧啶（嘌呤/嘧啶）序列。该嘌呤-嘧啶序列在大鼠、小鼠和人类AR基因启动子的相同近端位置保守。该区域的突变导致启动子活性下降3倍，表明其具有重要的调节功能。用单链特异性S1核酸酶检测AR嘌呤/嘧啶区域的构象状态，结果显示它能够形成一种涉及未配对单链的非B型DNA结构。对S1敏感位点的精细定位揭示了一种不对称切割模式，表明存在分子内三螺旋H型DNA构象。电泳迁移率变动分析表明，AR启动子的嘌呤/嘧啶区域能结合一种新型嘧啶单链特异性蛋白（ssPyrBF）以及一种双链DNA结合蛋白。寡核苷酸交叉竞争实验和抗体超迁移实验均证实，双链结合蛋白等同于Sp1。脱氧核糖核酸酶I（DNase I）足迹分析显示，Sp1与嘌呤/嘧啶位点有多个结合位点，与相邻的GC盒相比，Sp1与该区域的相互作用较弱，而Sp1在GC盒发挥作用以募集TFIID复合物。这些结果表明，当AR基因的嘌呤/嘧啶结构域以双链B-DNA构象存在时，它可用于吸引额外的Sp1分子。然而，ssPyrBF的结合以及由此导致的非B型DNA结构的稳定预计会阻止其与Sp1的相互作用。我们推测，在无TATA的AR基因启动子中，紧邻GC盒的嘌呤/嘧啶区域的多个弱Sp1位点可为功能性GC盒提供该转录因子的现成来源，从而促进起始复合物的组装。
