Gene structure and Function of H^+-ATPase.

为了研究ATP合成酶分子与质子易位耦合的ATP合成的分子机制，来自嗜热细菌的ATP合成酶具有很大的分析优势，因为该蛋白非常稳定，对各种实验处理都具有抗性。其次，对于细胞水平上基因表达调控的研究，人类培养的细胞是很好的靶点，因为已经建立了很多细胞系，并且已经报道了一些线粒体疾病。因此，我们已经采取了一些方法来了解ATP合成的分子机制和细胞对ATP形成的反应。(A) ATP合酶的分子机制。1)从嗜热细菌PS3中克隆ATP合酶操纵子基因并测序。2)在大肠杆菌中引入与强大肠杆菌相关的嗜热基因，可以使α、bata和γ亚基过表达。杆菌启动子。在纯化过程中，单个亚基被纯化而不使亚基变性。3)过表达的亚基可以被重组。上述温和的纯化程序使α和β亚基得以重构。4)通过定点诱变的方法，将亚基中的部分氨基酸人工替换为其他氨基酸。突变亚基的结合为分子机制提供了一些信息。过表达亚基被用于一些物理化学方法，包括中子散射和蛋白质的结晶。(B)人ATP合酶的细胞生物学。1)对编码ATP合成酶bata-亚基等7种线粒体蛋白的人cdna进行克隆测序。2)克隆并测序了人ATP合成酶β亚基和人丙酮酸脱氢酶α亚基的基因组基因。3)通过启动子活性分析5'上游区域。这些实验揭示了一种新的增强子的存在，这种增强子可以增强转录。此外，某些线粒体蛋白的增强子在某些基因中是共同的。4)通过Northern blotting检测不同细胞条件下基因表达的调控。5)从线粒体疾病患者中建立氧化磷酸化缺陷细胞系。少

For investigating the molecular mechanism of ATP synthesis on an ATP synthase molecule coupled with the proton translocation, ATP synthase from a thermophilic bacterium has a great advantage to be analyzed, because the protein is extremely stable and resistant against various kinds of experimental treatments. Next, for the studies on the regulation of the gene expression in the cellular level, the human cultured cells are good targets because a lot of cell-lines have been established and because some mitochondrial diseases have been reported. Therefore, we have taken approaches to understand the molecular mechanisms for ATP synthesis and the cellular response against ATP formation, respectively.(A) For molecular mechanism of ATP synthase. 1) The gene for the ATP synthase operon was cloned and sequenced from a thermophilic bacterium PS3. 2) The alpha ,bata and gamma subunits could be over-expressed in E. coli by introducing the thermophilic genes which had been connected with a strong E … More . coli promoter. The individual aubunits were purified without denaturating the subunits during the purification procedure. 3) The over-expressed subunit could be reconstituted. The above mentioned mild purification procedure enables the reconstitution of the alpha and bata subunits. 4) By site-directed mutagenesis method, some amino acid in the subunits were substituted with the other amino acids artificially. The combination of the mutated subunits gave several information on the molecular mechanisms. 5) The over-expressed subunits were used for some physico-chemical approaches including neutron scattering, and crystallization of the proteins.(B) The cell biology of the human ATP synthase. 1) The human cDNAs encoding 7 mitochondrial proteins including ATP synthase bata-subunit were cloned sequenced. 2) The genomic genes for the human ATP synthase bata-subunit and the human pyruvate dehydrogenase alpha-subunit were also cloned and sequenced. 3) The 5' upstream region were analyzed by the promoter activity. These kinds experiments revealed the presence of a novel enhancer which enhances the transcription. In addition, the enhancer were common to some genes for some mitochondrial proteins. 4) The regulation of the gene expression were examined by Northern blotting in the various kind of cellular conditions. 5) The oxidative phosphorylation-deficient cell lines were established from patients with mitochondrial disease. Less