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

该项目的总体目标是表征固氮、兼性异养蓝藻Nostoc punctiforme中葡萄糖-6-磷酸脱氢酶（G6 PD）表达的转录和翻译后调节。 G6 PD是氧化戊糖磷酸途径的起始酶。 本实验室的前期工作证实，G6 PD对固氮和氮素的暗生长是必不可少的。punctiforme，G6 PD的转录增加在生长过程中，在铵和N2的存在下，和G6 PD催化活性是依赖于一个共转录的基因称为opcA的产品。 具体目标是：1. 确定G6 PD转录调控机制与氮响应操作子/启动子区P1和P5的关系。 通过体内（报告基因表达）和体外（足迹法和径流转录测定）实验验证P1和P5的5'区中的启动子活性。 P1和P5的调节5'区将用于迁移率变动测定，以鉴定和分离起激活剂或阻遏剂作用的DNA结合蛋白。 2. 确定OpcA在G6 PD催化活性中的作用。 天然蛋白凝胶中的比活性、活性染色和免疫检测的测定将用于检查野生型和opcA突变菌株提取物中依赖于OpcA的G6 PD的催化和聚集状态。 这项工作将提供有关生态重要生物体转录的信息。 该项目的总体目标是确定参与葡萄糖-6-磷酸脱氢酶（G6 PD）在细胞中表达的生化机制。 蓝细菌（蓝绿色）Nostoc punctiforme。 实验室先前的工作确定，G6 PD对于这种生物和相关生物的固氮是必不可少的。 光合、固氮蓝藻是地球上最不依赖营养的生物，在光照环境中广泛分布，对维持环境稳定具有重要意义，可用于某些污染生境的生物修复。 调控G6 PD等酶合成的DNA序列可用于构建蓝藻生物降解代谢途径。 本项目有两个具体目标：鉴定在酶合成调控中重要的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鉴定G6 PD合成中的重要DNA序列和调节步骤。 通过同时测量酶活性和分子大小（聚集状态）来确定相关蛋白在形成更具活性的高度聚集状态的G6 PD中的作用。 该项目将提供关于一组具有生态意义的生物体的新信息。

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.