REGULATION OF THE YEAST PH05 GENE

这项工作的长期目标是了解 转录起始以及转录活性如何 调节子受信号转导途径的影响。对象的基因 在这些研究中使用的是酵母基因PHO5，它编码一种分泌的 一种酸性磷酸酶，其转录在高水平时受到严格抑制。 浓度的磷酸盐和诱导约1000倍， 对磷酸盐饥饿的反应在诱导后，PHO5启动子 在染色质结构上发生了巨大的变化， 破坏四个定位的核小体。一些分子 参与PHO5调节的基因已经通过遗传分析鉴定， 属于高等真核生物中高度保守的家族。这些 包括同源结构域蛋白PHO2、碱性螺旋环螺旋蛋白 PHO4和细胞周期蛋白-cdk（细胞周期蛋白依赖性激酶）复合物， PHO80和PHO85。 本提案的三个目标是：（1）确定 正转录调节因子PHO2和PHO4的作用，以及 确定他们的活动是如何被控制的。 (2)描述活动的特征 负调节因子PHO80和PHO85组成了一种蛋白质， 激酶，并确定其活性如何调节。(3)探讨 在PHO5启动子处的染色质转换的要求。一个 了解这些分子如何发挥作用并合作调节 PHO5将揭示信号转导之间的基本联系 途径和转录控制，也将提供深入了解 高等真核生物中的这些联系。PHO80-PHO85复合物代表 一个独特的机会来研究细胞周期蛋白依赖的功能， 激酶复合物：在这种情况下，生物学相关底物（PHO4） 已经鉴定，磷酸化的作用是已知的，可以 体外复溶。最后，对转录因子的研究表明， PHO5的控制可能有助于理解染色质的作用 基因调控中的结构。

The long-term goal of this work is to understand the regulation of transcription initiation and how the activity of transcriptional regulators is influenced by signal transduction pathways. The gene to be used in these studies is the yeast gene, PHO5, which encodes a secreted acid phosphatase whose transcription is tightly repressed in high concentrations of phosphate and induced approximately 1000-fold in response to phosphate starvation. Upon induction, the PHO5 promoter undergoes a dramatic alteration in chromatin structure that results in the disruption of four positioned nucleosomes. Several of the molecules involved in PHO5 regulation have been identified by genetic analysis and belong to highly conserved families found in higher eukaryotes. These include the homeodomain protein PHO2, the basic-helix-loop-helix protein PHO4, and the cyclin-cdk (cyclin-dependent kinase) complex consisting of PHO80 and PHO85. The three aims of this proposal are to: (1) Determine the mechanism of action of the positive transcriptional regulators, PHO2 and PHO4, and determine how their activity is controlled. (2) Characterize the activity of the negative regulators PHO80 and PHO85, which make up a protein kinase, and determine how its activity is regulated. (3) Investigate the requirements for the chromatin transition at the PHO5 promoter. An understanding of how these molecules function and cooperate to regulate PHO5 will reveal fundamental connections between signal transduction pathways and transcriptional control and will also provide insight into these connections in higher eukaryotes. The PHO80-PHO85 complex represents a unique opportunity to study the function of a cyclin-cyclin dependent kinase complex: in this case, a biologically relevant substrate (PHO4) has been identified, and the effect of phosphorylation is known and can be reconstituted m vitro. Finally, investigation of the transcriptional control of PHO5 may lead to an understanding of the role of chromatin structure in gene regulation.