Kang T, Martins T, Sadowski I
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1993 May 5;268(13):9629-35.
Transcription of the genes required for utilization of galactose in Saccharomyces cerevisiae is controlled primarily by the transcriptional activator protein GAL4. The upstream activating sequences for galactose (UASG) of most GAL genes have multiple sites to which GAL4 can bind. In this report we compare the binding properties of wild type GAL4 and derivatives of GAL4 bearing the N-terminal DNA-binding domain to multiple DNA-binding sites in vitro. To produce wild type GAL4, we constructed a recombinant baculovirus for expression in insect cells. Recombinant wild type GAL4 was found to bind efficiently to an oligonucleotide containing a near-consensus 17-mer GAL4 DNA-binding site in electrophoretic mobility shift assays. Footprinting experiments revealed that wild type GAL4 binds cooperatively to the four GAL4 DNA-binding sites of the GAL1-10 UASG; however, in contrast an N-terminal fragment of GAL4 containing only the DNA-binding/dimerization domains binds to each of these sites with slightly different affinity. With increasing concentrations of GAL4(1-147), the four sites become filled in the following order: site II, site IV, site I, and site III. In experiments with wild type GAL4, these four sites become fully occupied at approximately the same concentration of protein. In footprints of wild type GAL4 on the USAG, enhancements and protections of DNase I-sensitive cleavages are detectable between sites III and IV, indicative of formation of a loop between these distantly spaced sites. Binding of wild type GAL4 to a strong near-consensus binding site assists binding to an adjacent mutant site in both electrophoretic mobility shift and footprinting assays. GAL4(1-147) and GAL4(1-147) fused to portions of GAL4's activating region II were incapable of cooperative DNA binding in our assays. We conclude from these observations that wild type GAL4 has a cooperative DNA-binding function that is distinct from the DNA binding and dimerization or transcriptional activation functions, and likely plays and important role in precise regulation of GAL gene transcription.
酿酒酵母中利用半乳糖所需基因的转录主要受转录激活蛋白GAL4的控制。大多数GAL基因的半乳糖上游激活序列（UASG）有多个GAL4能结合的位点。在本报告中，我们在体外比较了野生型GAL4及其带有N端DNA结合结构域的衍生物与多个DNA结合位点的结合特性。为了产生野生型GAL4，我们构建了一种用于在昆虫细胞中表达的重组杆状病毒。在电泳迁移率变动分析中，发现重组野生型GAL4能有效地结合到含有接近一致的17聚体GAL4 DNA结合位点的寡核苷酸上。足迹实验表明，野生型GAL4能协同结合到GAL1 - 10 UASG的四个GAL4 DNA结合位点；然而，相比之下，仅包含DNA结合/二聚化结构域的GAL4 N端片段与这些位点中的每一个结合时亲和力略有不同。随着GAL4(1 - 147)浓度的增加，四个位点按以下顺序被占据：位点II、位点IV、位点I和位点III。在野生型GAL4的实验中，这四个位点在大约相同的蛋白质浓度下被完全占据。在野生型GAL4对USAG的足迹实验中，在位点III和IV之间可检测到DNase I敏感切割的增强和保护，这表明在这些远距离位点之间形成了一个环。在电泳迁移率变动分析和足迹实验中，野生型GAL4与一个强的接近一致的结合位点的结合有助于其与相邻突变位点的结合。在我们的实验中，GAL4(1 - 147)以及与GAL4激活区域II部分融合的GAL4(1 - 147)都不能进行协同DNA结合。我们从这些观察结果得出结论，野生型GAL4具有一种协同DNA结合功能，该功能不同于DNA结合、二聚化或转录激活功能，并且可能在GAL基因转录的精确调控中起重要作用。
