GENETIC REGULATION OF ALCOHOL METABOLISM IN YEAST

其目的是了解真核基因的调控。 核酸-蛋白质相互作用与效应物的代谢调节 分子。更具体地说，我们将确定蛋白质和DNA 赋予锌指特异性和高亲和力的成分 蛋白质与DNA的相互作用。这将通过分析DNA来实现 含有单一氨基酸变化的突变蛋白的结合特性 在ADR1的两个锌指中。突变体表现出的变化 当用突变的结合位点进行测试时，特异性将允许预测 将用于指导构建新的锌指蛋白 具有独特的DNA结合特性。通过改变相同的 相似蛋白质中的手指每个手指对 结合能可以计算出来。蛋白激酶A是否以及如何 ADR1的磷酸化影响其转录激活功能 将以多种方式确定。例如，站点和 ADR1磷酸化的化学计量比将被测定，并能力 磷酸化和非磷酸化的ADR1刺激转录 将在体外进行测量。ADH2激活的ADR1非依赖性途径 将通过识别通过以下方式唯一交互的突变体来表征 这条路。对依赖ADR1的突变体的进一步分析 ADH2的葡萄糖抑制将集中在可能揭示 ADR1依赖和非ADR1途径之间的相互作用 基因激活和抑制..通过创建新的DNA结合蛋白 可预测的绑定属性可能会更改 以令人满意的方式表达基因。因为人类的几种疾病 被确认是由于锌指转录因子的突变， 了解这些因素是如何发挥作用的，有助于理解 这些疾病的缺陷。

The objective is to understand the regulation of a eukaryotic gene in terms of nucleic acid-protein interactions and metabolic regulation of effector molecules. More specifically we will determine the protein and DNA components that impart specificity and high affinity to zinc finger protein-DNA interactions. This will be achieved by analyzing the DNA binding properties of mutant proteins containing single amino acid changes in the two zinc fingers of ADR1. Mutants which show a change of specificity when tested with mutated binding sites will allow predictions to be made that will guide the construction of new zinc finger proteins with unique DNA binding properties. By varying the number of identical fingers in similar proteins the contribution each finger makes to the energy of binding can be calculated. Whether, and how, protein kinase A phosphorylation of ADR1 influences its transcription activation function will be determined in a variety of ways. For example, the sites and stoichiometry of phosphorylation of ADR1 will be determined and the ability of phosphorylated and nonphosphorylated ADR1 to stimulate transcription in vitro will be measured. The ADR1-independent pathway of ADH2 activation will be characterized by identifying mutants that interact uniquely through this pathway. Further analysis of ADR1-dependent mutants that alleviate glucose repression of ADH2 will focus on mutants that might reveal interactions between the ADR1-dependent and ADR1-independent pathways of gene activation and repression.. By creating new DNA binding protein with predictable binding properties it might be possible to alter the level of gene expression in desirable ways. Since several human diseases have been identified that are due to mutations in zinc finger transcription factors, understanding how these factors function is useful to an understanding of the defect in these diseases.