CU-REGULATED GENE EXPRESSION & METALLOPROTEIN SYNTHESIS

金属离子调控基因机制的研究进展 表达和金属蛋白的生物合成/组装在这里被提出。这个 该项目的重点是两种蛋白质，质蓝蛋白和细胞色素C6，这两种蛋白质 光合作用电子转移催化剂。我们已经证明了 这些蛋白质的生物合成受制于金属感应调节。 系统中，铜在纳摩尔浓度下是引发 作为回应，一种蛋白质(含铜的)的积累 )和对另一种(功能相同的血红素)的抑制 蛋白CytC6)。分子的详细表征 这一依赖铜的调控系统的组成和运行模式 是我们工作的目标。我们将确定铜监管要素 细胞色素C6衣原体基因(Cres)体内缺失分析 并识别序列特异性DNA结合蛋白(BP)，其功能是 铜依赖的体外转录调控因子。建议的角色是 通过对菌株的研究，将证实叶绿体花青素是一种“铜汇” 含有减少或增加的全叶绿色素的。为了获得一份 更完整和全面的监管电路图景，我们将 分离并鉴定显示细胞色素C6调节改变突变体， 我们将研究另外两个铜反应的调控特征 蛋白质--栅栏藻相互调节的胞质蓝蛋白，以及一个 衣藻30 kDa蛋白的协同调控。邮报- 叶绿体蛋白和cytc6成熟的翻译途径是 通过分子和基因解剖进行研究。而且，我们将发起一项 藻蓝蛋白和细胞色素C6的结构比较--结构上的差异 但在功能上等同的电子转移催化剂。 过渡金属是生物系统的重要组成部分。通过 阐明它们的用途和功能(作为催化剂和调节剂)， 我们的工作有助于对基础生物化学的理解 在金属代谢方面的加工和实际应用 以疾病为基础的疾病和金属的潜在创造/修饰- 抵抗力/耐受力。 我的长期目标是让自己成为一名独立的调查员 金属蛋白的生物合成、结构和功能领域。在 在接下来的五年里，我将继续推进上述项目并加强研究 将遗传和结构结合起来的梅实验室的能力 对我们的项目进行研究。结合已建立的分子和 生化技术，这些新的方法将扩大我们的实验 为调查奠定了基础，开辟了新的调查途径。虽然我的知识分子 遗传和结构背景很强，我需要获得实用知识 在这些相关领域积累经验并加强我的研究专长 纪律。一个RCDA，考虑到我密集的研究参与， 为发展新领域的经验和技能提供时间将 大大促进了我事业的发展。 化学和生物化学系提供了大量的 我的研究项目所需的空间、资金和设施。该部门充分 支持RCDA申请，以促进我的研究事业 把我从所有本科教学任务中解脱出来 研究生(与研究无关)“核心”教学职责。

A research program concerned with the mechanism of metal ion regulated gene expression and metalloprotein biosynthesis/assembly is proposed here. The project focusses on two proteins, plastocyanin and cytochrome c6 that are photosynthetic electron transfer catalysts. We have shown that the biosynthesis of these proteins is subject to a metal-sensing regulatory system where Cu, at nanomolar concentrations, is the signal which elicits as a response the accumulation of one protein (the Cu-containing plastocyanin) and the repression of the other (functionally equivalent heme protein cyt c6). The detailed characterization of the molecular constituents and mode of operation of this Cu-dependent regulatory system is the objective of our work. We will identify copper-regulatory elements (CREs) in the Chlamydomonas gene for cyt c6 by in vivo deletion analysis and identify sequence-specific DNA binding proteins (BPs) that function as Cu-dependent transcriptional regulators in vitro. The proposed role of plastocyanin as a "Cu-sink" will be verified by the study of strains containing decreased or increased levels of holoplastocyanin. To gain a more complete and comprehensive picture of regulatory circuits, we will isolate and characterize mutants that display altered regulation of cyt c6, and we will examine the regulatory features of two other Cu-responsive proteins -- the reciprocally regulated plastocyanin of Scenedesmus, and a coordinately regulated 30 kDa protein of Chlamydomonas. The post- translational pathway for maturation of plastocyanin and cyt c6 will be investigated by molecular and genetic dissection. And, we will initiate a structural comparison of plastocyanin and cyt c6 -- structurally distinct but functionally equivalent electron transfer catalysts. Transition metals are essential components of biological systems. By elucidating their utilization and function (as catalysts and regulators), our work contributes to the understanding of fundamental biochemical processes and has practical applications with respect to metal-metabolism based diseases and the potential creation/modification of metal- resistance/tolerance. My long-term goal is to establish myself as an independent investigator in the area of metalloprotein biosynthesis, structure and function. In the next five years, I will pursue the above projects and enhance the research capabilities of may laboratory by incorporating genetic and structural studies into our program. In combination with established molecular and biochemical techniques, these new approaches will expand our experimental base and lead to new avenues of investigation. Although my intellectual background in genetics and structure is strong, I need to acquire practical experience and strengthen my research expertise in these relevant disciplines. An RCDA, in allowing for my intensive research involvement, and providing time for developing experience and skills in new areas would significantly promote the development of my career. The department of Chemistry and Biochemistry has provided substantial space, funds and facilities for my research program. The department fully supports the application for an RCDA in order to promote my research career and will release me from all undergraduate teaching assignments and from graduate (non-research related) "core" teaching responsibilities.