Regulation of Glucose-6-Phosphate Dehydrogenase Expression in Nostoc Punctiforme

发菜中葡萄糖-6-磷酸脱氢酶表达的调控

• 批准号: 9604270
• 负责人: John Meeks
• 金额: $ 30万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604270&HistoricalAwards=false
• 关键词: Regulation; Glucose; Phosphate; Dehydrogenase; Expression

9604270 Meeks The overall goal of this project is to characterize transcriptional and posttranslational regulation of glucose-6-phosphate dehydrogenase (G6PD) expression in the nitrogen fixing, facultatively heterotrophic cyanobacterium Nostoc punctiforme. G6PD is the initial enzyme of the oxidative pentose phosphate pathway. Previous work in this laboratory established that G6PD is essential for nitrogen fixation and dark growth of N. punctiforme, that transcription of G6PD increases during growth in the absence of ammonium and presence of N2, and that G6PD catalytic activity is dependent on the product of a cotranscribed gene termed opcA. The specific objectives are to: 1. Identify the mechanisms of regulation of transcription of G6PD with the respect to the nitrogen responsive operator/promoter region P1 and P5. Promoter activity in the 5' regions of P1 and P5 will be verified by in vivo (reporter gene expression) and in vitro (footprinting and runoff transcription assay) experiments. The regulatory 5' regions of P1 and P5 will be used in mobility shift assays to identify and isolate DNA binding proteins that function as activators or repressors. 2. Determine the role of OpcA in G6PD catalytic activity. Assays of specific activity, and activity stains and immunodetection in native protein gels will be used to examine the catalytic and aggregation states of G6PD, dependent on OpcA, in extracts from wild-type and opcA- mutant strains. This work will provide information about transcription in an ecologically significant organism. The overall goal of this project is to identify the biochemical mechanisms involved in expression of the enzyme glucose-6-phosphate dehydrogenase (G6PD) in the cyanobacterium (blue-green alga) Nostoc punctiforme. Previous work in the laboratory established that G6PD is essential for nitrogen fixation in this, and related, organisms. Photosynthetic, nitrogen fixing cyanobacteria are the most nutritionally independent organisms on earth and have an es sentially ubiquitous distribution in habitats exposed to light; thus, they are important for maintaining stable environments might be used in bioremediation of certain polluted habitats. DNA sequences that regulate the synthesis of enzymes such as G6PD could be used to construct biodegrading metabolic pathways in cyanobacteria. There are two specific objectives in this project: identification of DNA sequence important in regulation of enzyme synthesis, and of protein-protein interactions in enzyme activity. Biochemical assays monitoring protein-DNA interactions will be used to identify the important DNA sequences and regulated steps in the synthesis of G6PD. The role of an associated protein in the formation of more active highly aggregated states of G6PD will be determined by concurrently measuring enzyme activity and molecular size (aggregated state). This project will provide new information about an ecologically significant group of organisms.

9604270 Meeks该项目的总体目标是表征氮固定中葡萄糖-6-磷酸脱氢酶（G6PD）表达的转录和翻译后调节，在夫作上，夫作杂质核细菌Nostoc Punctiforne。 G6PD是氧化五五磷酸途径的初始酶。 该实验室的先前工作表明，G6PD对于氮的固定氮和深色生长至关重要，在不存在铵和N2的情况下，G6PD的转录在生长过程中增加，而G6PD催化活性取决于cotranscranscranscranscransed Gene perned opca的产物。 具体目标是：1。确定G6PD转录的调节机制，并与氮响应型操作员/启动子区域P1和P5相关。 P1和P5的5'区域中的启动子活性将通过体内（报告基因表达）和体外（足迹和径流转录测定）实验来验证。 P1和P5的调节5'区域将用于迁移率转移测定中，以识别和分离起活化剂或阻遏物的DNA结合蛋白。 2。确定OPCA在G6PD催化活性中的作用。 在野生型和opca突变菌株中提取的提取物中，对天然蛋白质凝胶的特定活性以及活性染色和免疫检测的测定将用于检查G6PD的催化和聚集态。 这项工作将提供有关生态意义上有机体中转录的信息。 该项目的总体目标是确定葡萄糖-6-磷酸脱氢酶（G6PD）在蓝细菌（蓝绿色藻类）Nostoc Punctiform中所涉及的生化机制。 实验室中的先前工作表明，G6PD对于氮和相关生物的氮固定至关重要。 光合作用，氮固定蓝细菌是地球上营养最独立的生物，在暴露于光线的栖息地中具有eS子无处不在的分布。因此，它们对于维持稳定环境很重要，可以用于对某些污染栖息地进行生物修复。 调节酶（例如G6PD）合成的DNA序列可用于构建蓝细菌中的生物降解代谢途径。 该项目中有两个特定的目标：鉴定DNA序列在调节酶合成的重要性以及酶活性中蛋白质 - 蛋白质相互作用的鉴定。 监测蛋白-DNA相互作用的生化测定将用于识别重要的DNA序列和调节G6PD合成的步骤。 相关蛋白在形成更活跃的G6PD状态中的作用将通过同时测量酶活性和分子大小（聚集态）来确定。 该项目将提供有关生态意义的生物群体的新信息。