REGULATION OF THE YEAST PH05 GENE

这项工作的长期目标是理解 转录起始和转录活性如何 调节因子受信号转导途径的影响。未来的基因是 在这些研究中使用的是酵母基因pho5，它编码一种分泌的 转录在高密度脂蛋白中被严格抑制的酸性磷酸酶 磷酸盐浓度和诱导的大约1000倍于 对磷酸盐饥饿的反应。诱导后，PHO5启动子 染色质结构发生了戏剧性的变化，导致 四个定位的核小体破裂。其中几个分子 参与PHO5调节的基因已经通过遗传分析和 属于高等真核生物中发现的高度保守的家族。这些 包括同源域蛋白PHO2，即碱性-螺旋-环-螺旋蛋白 PHO4，以及由以下组成的细胞周期蛋白-cdk(细胞周期蛋白依赖激酶)复合体 PHO80和PHO85。 这项建议的三个目的是：(1)确定 正转录调节因子PHO2和PHO4的作用，以及 确定如何控制他们的活动。(2)描述活动的特征 负性调节因子PHO80和PHO85组成蛋白质 并确定其活性是如何被调节的。(3)调查 PHO5启动子染色质转换的要求。一个 了解这些分子如何发挥作用并协同调节 PHO5将揭示信号转导之间的基本联系 途径和转录调控，也将提供洞察 这些连接在高等真核生物中。PHO80-PHO85复合体代表 研究细胞周期蛋白依赖性细胞周期蛋白功能的难得机会 激酶复合体：在这种情况下，与生物相关的底物(PHO4)具有 已经确定，并且磷酸化的影响是已知的，并且可以 重组m体外培养。最后，对转录本进行了研究。 控制PHO5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.