EUKARYOTIC GENE REGULATION IN YEAST

这笔赠款的长期目标是进一步了解如何 真核转录调控因子与DNA结合并影响基因 抄写。尽管拟议的实验涉及转录 来自各种有机体的调节器，并解决各种问题， 所有这些都涉及，至少部分涉及在酵母中的操纵，以促进 实现目标。具体目标分为三大类。 首先，一系列实验涉及GAL4，A 被广泛研究的来自酵母的“通用”激活剂，激活转录。 将特别注意辅助剂GAL11的影响 以某种方式帮助GAL4和某些其他激活剂的因素。遗传和 生理学实验将探索GAL11形成复合体的想法 有了这些激活剂，可能涉及额外蛋白质的复合体 井。第二，GAL4家族成员的机制 识别DNA中特定位置的激活剂将被分析。这是一个 蛋白质DNA相互作用的特别有趣的例子：每一个 蛋白质(如GAL4、PUT3和PPR1)含有相似的“锌簇”元素 接触相同的DNA三联体；每个三联体的特征 DNA位点显然是这些被识别的三联体之间的间距。 在其他实验中，将分析嵌合蛋白以揭示 根据蛋白质DNA的特异性。第三，果蝇的行为 将对形态形成背部进行分析。推定的辅助因素可能是 将背部从激活剂转换为负调节器将是 克隆，就像被抑制的天然果蝇激活剂一样 背部工作与其推定的辅助因素相结合。结构性的 背部与仙人掌之间以及背部(A)之间相互作用的基础 REL类激活剂的成员)和DNA将被分析。

The long term goal of this grant is to further the understanding of how eukaryotic transcriptional regulators bind to DNA and affect gene transcription. Although the proposed experiments involve transcriptional regulators from a variety of organisms and address a variety of questions, all involve, at least in part, manipulations in yeast to facilitate achieving the goals. The specific aims fall into three broad categories. First, a series of experiments concern the mechanism by which GAL4, a widely studied "universal" activator from yeast, activates transcription. Particular attention will be paid to the effect of GAL11 an auxiliary factor that somehow helps GAL4 and certain other activators. Genetic and physiological experiments will probe the idea that GAL11 forms a complex with these activators, a complex that may involve additional proteins as well. Second, the mechanism by which members of the GAL4 family of activators recognize specific sites in DNA will be analyzed. This is a particularly intriguing example of protein DNA interaction: each of these proteins (eg GAL4, PUT3 and PPR1) bears similar "zinc cluster" elements that contact identical DNA triplets; the distinguishing feature of each DNA site is evidently the spacing between these recognized triplets. Among other experiments, chimeric proteins will be analyzed to reveal the basis of the protein DNA specificity. Third, the action of the Drosophila morphogen Dorsal will be analyzed. The putative co-factor that presumably converts Dorsal from an activator into a negative regulator will be cloned, as will the natural Drosophila activator that is inhibited by Dorsal working in conjunction with its putative co-factor. The structural basis for the interaction between Dorsal and Cactus, and between Dorsal (a member of the rel class of activators) and DNA will be analyzed.