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

该项目的总体目标是表征葡萄糖-6-磷酸脱氢酶（G6PD）在固氮兼性异养蓝藻Nostoc punctiformme中表达的转录和翻译后调控。G6PD是氧化戊糖磷酸途径的起始酶。本实验室之前的研究证实，G6PD对N.点状霉的固氮和暗生长至关重要，G6PD的转录在缺乏铵和N2的情况下会增加，G6PD的催化活性依赖于一个共转录基因opcA的产物。具体目标是：1。确定G6PD在氮响应算子/启动子区P1和P5方面的转录调控机制。P1和P5的5'区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的调控5'区将用于迁移转移试验，以鉴定和分离作为激活或抑制功能的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突变菌株的提取物中，将使用特异性活性、活性染色和免疫检测来检测依赖于OpcA的G6PD的催化和聚集状态。这项工作将提供有关转录在生态重要的生物体的信息。本项目的总体目标是确定葡萄糖-6-磷酸脱氢酶（G6PD）在蓝藻（蓝绿藻）Nostoc punctiformme中表达的生化机制。先前在实验室的研究证实，G6PD对这种生物及相关生物的固氮至关重要。光合作用、固氮的蓝藻是地球上最具营养独立性的生物，在光照环境中几乎无处不在；因此，它们对维持稳定的环境很重要，可能用于某些受污染生境的生物修复。调控G6PD等酶合成的DNA序列可用于构建蓝藻生物降解代谢途径。在这个项目中有两个特定的目标：鉴定在酶合成调控中重要的DNA序列，以及酶活性中的蛋白质-蛋白质相互作用。监测蛋白质-DNA相互作用的生化分析将用于鉴定重要的DNA序列和G6PD合成中的调节步骤。相关蛋白在G6PD形成更活跃的高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。这个项目将提供一个具有重要生态意义的生物群的新信息。

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.