GENETIC REGULATION OF ALCOHOL METABOLISM IN YEAST

目的是了解真核基因的调控， 核酸-蛋白质相互作用和效应物的代谢调节 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组分 蛋白质-DNA相互作用 这将通过分析DNA 含有单个氨基酸变化的突变蛋白的结合特性 在ADR 1的两个锌指中 突变体表现出 当用突变的结合位点测试时，特异性将允许预测 这将指导新的锌指蛋白的构建 具有独特的DNA结合特性 通过改变相同的 类似蛋白质中的手指，每个手指对蛋白质的贡献 可以计算结合能。 蛋白激酶A是否以及如何 ADR 1的磷酸化影响其转录激活功能 将以多种方式决定。 例如，网站和 将确定ADR 1磷酸化的化学计量， 磷酸化和非磷酸化的ADR 1刺激转录， 将测量体外。 ADR 1非依赖性ADH 2激活途径 将通过识别突变体来表征， 这条路。 进一步分析缓解ADR 1依赖性突变体 葡萄糖抑制ADH 2将集中在突变体，可能揭示 ADR 1依赖性和ADR 1非依赖性途径之间的相互作用 基因激活和抑制通过创造新的DNA结合蛋白， 可预测的绑定属性，可以改变 基因表达的方式。 自从几种人类疾病 鉴定为由于锌指转录因子的突变， 了解这些因素如何起作用有助于了解 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.