Jarvis E E, Clark K L, Sprague G F
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Genes Dev. 1989 Jul;3(7):936-45. doi: 10.1101/gad.3.7.936.
Two proteins, alpha 1 and pheromone/receptor transcription factor (PRTF), bind cooperatively to the upstream activation sequences (UAS) of yeast alpha-specific genes and thereby activate their transcription. In these protein-DNA complexes, the PRTF moiety interacts with a degenerate dyad symmetric sequence, the P box. PRTF contributes also to the regulation of a second set of cell-type-specific genes, the a-specific genes. We used two in vitro assays to show that PRTF is encoded, at least in part, by the MCM1 gene. In one assay, truncated MCM1 proteins encoded by deletion derivatives of the MCM1 gene formed protein-DNA complexes of novel mobility, demonstrated that MCM1 can bind to the P-box-containing DNA. Second, antibodies raised to a synthetic MCM1 polypeptide retard the migration of PRTF-DNA complexes in gel mobility shift assays. This result indicates that PRTF, defined as an activity that binds cooperatively with alpha 1 to alpha-specific UAS elements, shares an epitope with MCM1. In addition, we show that MCM1 deletions that remove the carboxy-terminal 129 codons of 286 total codons encode truncated MCM1 molecules that are competent to activate transcription in vivo, indicating that the carboxy-terminal residues are not required for this process.
两种蛋白质,α1和信息素/受体转录因子(PRTF),协同结合到酵母α特异性基因的上游激活序列(UAS)上,从而激活它们的转录。在这些蛋白质 - DNA复合物中,PRTF部分与一个简并的二元对称序列P盒相互作用。PRTF还参与调控另一组细胞类型特异性基因,即a特异性基因。我们使用两种体外试验来表明PRTF至少部分由MCM1基因编码。在一种试验中,由MCM1基因的缺失衍生物编码的截短MCM1蛋白形成了具有新迁移率的蛋白质 - DNA复合物,证明MCM1可以结合含P盒的DNA。其次,针对合成MCM1多肽产生的抗体在凝胶迁移率变动分析中减缓了PRTF - DNA复合物的迁移。这一结果表明,被定义为与α1协同结合到α特异性UAS元件的活性的PRTF与MCM1共享一个表位。此外,我们表明,去除总共286个密码子的羧基末端129个密码子的MCM1缺失编码了在体内能够激活转录的截短MCM1分子,这表明该过程不需要羧基末端残基。