GENETIC STUDIES OF GLUTAMINE SYNTHETASE IN BACTERIA

基因ntrA、ntrB和ntrC的产物是 氮调节的转录在肠道细菌。 产品 ntrA基因的编码区（称为NTRA）是RNA的交替sigma亚基， 聚合酶：NTRA加核心RNA聚合酶（称为“核心RNA聚合酶”） NTRA全酶）可以结合到一个“强”的氮调节 在没有其他因素的情况下启动子。 ntrC产品 （称为NTRC），这是一种DNA结合蛋白， 通过NTRA全酶有效启动转录。 两个氮调节启动子中NTRC的结合位点 区域位于启动子上游100- 150 bp（76，101）， 似乎类似于真核生物中的转录增强子（76）。 为了激活转录，NTRC必须被磷酸化。 是 被ntrB的产物磷酸化和去磷酸化 基因（NTRB），这是一个双功能蛋白激酶- 磷蛋白磷酸酶（69，50）。 磷酸化程度 NTRC是受代谢调节的。 代谢调控 控制双功能酶NTRB的活性， 涉及至少两种额外的蛋白质，其中一种似乎 是细胞氮营养状况的主要传感器。 我们将进行生物化学研究和结构-功能 分析NTRC，以确定精确的机制， 这种增强子激活蛋白刺激启动 转录。 我们还将进行结构-功能分析 的sigma因子NTRA，以确定其 与NTRC、核心聚合酶和启动子DNA的相互作用。 这些研究将得到促进的事实，NTRA是不是 对细胞活力至关重要。 最后，我们将详细研究 其他蛋白质对NTRB活性的代谢控制的性质 和小分子。 我们在这方面的最终目标是 确定代谢物谷氨酰胺和2- 酮戊二酸与NTC转录速率直接相关 和NTRA全酶，如果是这样的话， 尺寸必须不同，以提供完整的生理范围， 转录率。

The products of genes ntrA, ntrB, and ntrC are required for nitrogen-regulated transcription in enteric bacteria. The product of gene ntrA (called NTRA) is an alternate sigma subunit for RNA polymerase: NTRA plus core RNA polymerase (referred to as NTRA holoenzyme) can bind to a "strong" nitrogen-regulated promoter in the absence of other factors. The ntrC product (called NTRC), which is a DNA binding protein, is required for efficient initiation of transcription by NTRA holoenzyme. Binding sites for NTRC in two nitrogen-regulated promoter regions lie 100-150bp upstream of the promoter (76,101) and appear to resemble transcriptional enhancers in eukaryotes (76). To activate transcription NTRC must be phosphorylated. It is phosphorylated--and dephosphorylated--by the product of the ntrB gene (NTRB), which is a bifunctional protein kinase- phosphoprotein phosphatase (69,50). The degee of phosphorylation of NTRC is metabolically regulated. Metabolic regulation controls the activities of the bifunctional enzyme NTRB and involves at least two additional proteins, one of which appears to be a primary sensor of the status of cellular nitrogen nutrition. We will undertake biochemical studies and a structure-function analysis of NTRC in order to determine the precise mechanism by which this enhancer activating protein stimulates initiation of transcription. We will also perform a structure-function analysis of the sigma factor NTRA to determine the nature of its interactions with NTRC, core polymerase and promoter DNA. These studies will be facilitated by the fact that NTRA is not essential for cell viability. Finally, we will study in detail the nature of metabolic controls of NTRB activity by other proteins and by small molecules. Our eventual goal in this regard is to determine whether pool sizes of the metabolites glutamine and 2- oxoglutarate correlate directly with rates of transcription by NTC and NTRA holoenzyme and, if so, the extent to which these pool sizes must vary to give the full physiological range of transcription rates.