GENETIC ANALYSIS AND FUNCTIONAL SITES

遗传分析和功能位点

• 批准号: 3294450
• 负责人: M. FEVZI DALDAL
• 金额: $ 17.68万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-12-01 至 1992-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294450
• 关键词: NADPH cytochrome c2 reductase; Rhodopseudomonas; bacterial genetics; chemical group; ferredoxin; gene expression; genetic manipulation; genetic mapping; genetic recombination; genetic strain; hydrogen transport; membrane potentials; membrane proteins; molecular cloning; mutant; nucleic acid sequence; oxidoreductase inhibitor; photosynthetic bacteria; spectrometry; structural genes

The ubiquinol:cytochrome c oxidoreductase (or the bc1 complex) is involved in the formation of the membrane potential and proton gradient, a vital process for living cells. The long term goal of this project is to understand, in molecular terms, how the structural subunits of the bc1 complex interact with their associated prosthetic groups to perform this role. As an initial step, the structural genes of the bc1 complex of Rhodopseudomonas capsulata, a photosynthetic prokaryote, have been isolated and their nucleotide sequences have been determined. The in vivo role of this complex has also been assessed by constructing null bc1 mutants. We now plan to undertake a detailed molecular genetic analysis to correlate the function sites of the bc1 complex (quinol oxidation, quinone reduction, and cytochrome c reduction) with its structural subunits (the FeS protein, cytochrome b, and cytochrome c1). For this purpose we will (1) select mutants resistant to specific inhibitors of the bc1 complex; (2) obtain new bc1 mutants that assemble a functionally altered complex; (3) map and sequence these mutations, including aer126 of R126, a mutant defective in the quinol oxidation site of the complex; and (4) change the amino acid residues thus defined to various other residues by sitedirected oligonucleotide mutagenesis to correlate the mutated sites with the perturbed functions. Insights gained in this prokaryotic system should be generally applicable to structurally more complex but functionally similar mitochondrial and chloroplast oxidoreductases of higher organisms, which are often refractory to genetic analysis. In general, this work should also contribute to a better understanding of functional interactions between the prosthetic groups and the structural subunits of membrane proteins.

泛醇：细胞色素 C 氧化还原酶（或 bc1 复合物） 参与膜电位和质子的形成 梯度，活细胞的一个重要过程。 长期目标是 这个项目旨在从分子角度理解如何 bc1复合物的结构亚基与其相互作用 相关的假肢组来执行此角色。 作为初始 步骤，bc1复合体的结构基因 荚膜红假单胞菌 (Rhodopseudomonas capsulata) 是一种能进行光合作用的原核生物，具有 已分离并确定其核苷酸序列 决定。 该复合物的体内作用也已被证实 通过构建无效 bc1 突变体进行评估。 我们现在计划 进行详细的分子遗传学分析以关联 bc1 复合物的功能位点（醌氧化、醌 还原和细胞色素 c 还原）及其结构 亚基（FeS 蛋白、细胞色素 b 和细胞色素 c1）。 为了 为此目的，我们将（1）选择对特定的抗性突变体 bc1复合物抑制剂； (2) 获得新的bc1突变体 组装一个功能改变的综合体； (3)图谱和序列 这些突变，包括 R126 的 aer126，这是一种缺陷突变体 配合物的对苯二酚氧化位点； (4)改变氨基 由此将酸残基定义为各种其他残基 定点寡核苷酸诱变以关联 具有扰动功能的突变位点。 在此获得的见解 原核系统应该普遍适用于结构 更复杂但功能相似的线粒体 高等生物的叶绿体氧化还原酶，通常 难以进行遗传分析。 总的来说，这项工作还应该 有助于更好地理解功能交互 假体基团和结构亚基之间 膜蛋白。