CURESPONSIVE TRANSCRIPTIONAL REGULATION

本申请中描述的研究计划涉及 铜反应基因表达的机制。实验 模型是绿色衣藻莱茵衣藻， 适用于铜及铜-蛋白质代谢的研究。 先前的研究表明，几种蛋白质的积累 在这种生物体中， 媒介其中之一是质体蓝素，一种丰富的铜- 含有光合电子转移催化剂，其铜- 依赖性积累受金属特异性全蛋白的影响 铜蛋白的形成和增加的稳定性 脱辅基蛋白另一种是细胞色素C6（一种含有血红素的 取代电子转移蛋白），其合成被诱导 在缺铜细胞中通过其基因的转录激活 介导的-铜响应元件（CuRE）和CuRE结合元件 蛋白质（BP）。第三种是粪卟啉原（粪原） 氧化酶是血红素生物合成后期的催化剂 途径，这是诱导协调与cytc 6在铜- 缺陷细胞 计划在下一个项目期间进行的研究的目标 是从生物化学的角度， 遗传学铜依赖性转录因子的组成部分 反应在这个系统中，并阐明分子基础 它的一些关键特性，特别是灵敏度和 金属调节剂相对于其金属的选择性 DNA结合位点。重点将放在CuRE-BP上， 将基于其序列特异性结合来纯化 先前鉴定的与cyt c6基因相关的CuRE。 将采用生物化学和生物物理分析来了解 其铜响应功能的结构基础。cDNA和 将通过以下方法克隆编码CuRE-BP的基因组序列： 常规方法。在平行实验中，“标记的”调节 突变体将通过筛选插入- 突变细胞的铜响应性表达的丧失 报告基因标记基因的野生型等位基因将被标记。 克隆和表达，这样就可以用反向遗传方法， 用于金属调节剂的功能分析。一 第二个目的是分离和分子表征 粪原氧化酶基因上的铜响应元件 目的是确定共同的调节途径是否 几个铜反应基因，如果是这样，是否CuRE-BP 在不同基因上显示CuRE的优先利用。 这项研究计划的长期目标是 了解真核细胞中的铜稳态， 特别是铜对基因表达的调节， 适应铜缺乏的环境。拟建项目 将扩大我们对反应机制的了解， 生物分配营养有限的资源， 维持新陈代谢的基本功能，这样做， 有助于了解人体铜不平衡， 由于摄入量减少、排泄过多或遗传 疾病

The research program described in this application deals with the mechanism of copper-responsive gene expression. The experimental model is the green alga Chlamydomonas reinhardtii which is ideally suited for studies of copper and copper-protein metabolism. Previous work has shown that the accumulation of several proteins in this organism is dependent upon the concentration of copper in the medium. One of these is plastocyanin, an abundant copper- containing photosynthetic electron transfer catalyst, whose copper- dependent accumulation is effected by metal-specific holoprotein formation and increased stability of the copper-protein relative to the apoprotein. Another is cytochrome C6 (a heme containing substitute electron transfer protein), whose synthesis is induced in copper-deficient cells by transcriptional activation of its gene mediated -a copper-responsive elements (CuREs) and a CuRE-binding protein (BP). A third is the enzyme coproporphyrinogen (coprogen) oxidase, a catalyst of a late step in the heme biosynthetic pathway, which is induced coordinately with cyt c6 in copper- deficient cells. The objective of the research planned for the next project period is to identify and characterize biochemically and by reverse genetics the components of the copper-dependent transcriptional response in this system, and to elucidate the molecular basis for some of its key features, particularly the sensitivity and selectivity of the metalloregulator with respect to its metal- and DNA-binding site(s). Emphasis will be placed on the CuRE-BP which will be purified on the basis of its sequence specific binding to previously identified CuREs associated with the cyt c6 gene. Biochemical and biophysical analyses will be employed to understand the structural basis of its copper-responsive function. cDNA and genomic sequences encoding the CuRE-BP will be cloned by conventional methods. In parallel experiments, "tagged' regulatory mutants will be identified by screening colonies of insertionally- mutagenized cells for loss of copper-responsive expression of a reporter gene. The wild-type alleles of the tagged genes will be cloned and expressed so that a reverse genetic approach can be exploited for the functional analysis of the metalloregulator. A second aim is the isolation and molecular characterization of copper responsive elements on the coprogen oxidase gene with the objective of determining whether a common regulatory pathway serves several copper-responsive genes and, if so, whether the CuRE-BP displays preferential utilization of CuREs on different genes. The long term objective of this research program is the understanding of copper homeostasis in eukaryotic cells, specifically the regulation of gene expression by copper in the context of adaptation to copper deficiency. The proposed project will expand our knowledge of the response mechanisms by which organisms allocate nutritionally-limited resources so as to maintain metabolically-essential functions, and in so doing, will contribute to the understanding of human copper imbalance which can result from reduced intake, excessive excretion or genetic diseases.