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固氮、兼性异养蓝细菌点状发菜中葡萄糖-6-磷酸脱氢酶 (G6PD) 表达的转录和翻译后调节。 G6PD 是氧化戊糖磷酸途径的起始酶。 该实验室之前的工作证实，G6PD 对于点状猪笼草的固氮和暗生长至关重要，在没有铵和存在 N2 的情况下，G6PD 的转录在生长过程中增加，并且 G6PD 催化活性依赖于称为 opcA 的共转录基因的产物。 具体目标是： 1. 确定与氮响应操纵基因/启动子区域 P1 和 P5 相关的 G6PD 转录调节机制。 P1 和 P5 5' 区域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）实验进行验证。 P1 和 P5 的 5' 调控区域将用于迁移率变化测定，以识别和分离充当激活剂或阻抑剂的 DNA 结合蛋白。 2.确定OpcA在G6PD催化活性中的作用。 天然蛋白质凝胶中的比活性测定、活性染色和免疫检测将用于检查野生型和 opcA 突变株提取物中依赖于 OpcA 的 G6PD 的催化和聚集状态。 这项工作将提供有关具有生态意义的生物体中转录的信息。 该项目的总体目标是确定蓝藻（蓝绿藻）发菜中葡萄糖-6-磷酸脱氢酶（G6PD）表达所涉及的生化机制。 实验室先前的工作证实，G6PD 对于该生物体及相关生物体的固氮至关重要。 进行光合作用、固氮的蓝细菌是地球上营养最独立的生物体，在暴露于光照的栖息地中基本上无处不在。因此，它们对于维持稳定的环境非常重要，可用于某些污染栖息地的生物修复。 调节 G6PD 等酶合成的 DNA 序列可用于构建蓝细菌中的生物降解代谢途径。 该项目有两个具体目标：鉴定在酶合成调节中重要的 DNA 序列，以及酶活性中蛋白质-蛋白质相互作用。 监测蛋白质-DNA 相互作用的生化测定将用于识别重要的 DNA 序列和 G6PD 合成中的调控步骤。 相关蛋白在形成更活跃的 G6PD 高度聚集状态中的作用将通过同时测量酶活性和分子大小（聚集状态）来确定。 该项目将提供有关具有重要生态意义的生物群的新信息。