Genetic and Biochemical Studies of the Tryptophan Operon

色氨酸操纵子的遗传和生化研究

• 批准号: 9206321
• 负责人: Charles Yanofsky
• 金额: $ 73万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-15 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9206321&HistoricalAwards=false
• 关键词: Genetic; Biochemical; Studies; Tryptophan; Operon

Studies will continue on the regulatory-metabolic network that modulates uptake, synthesis and utilization of tryptophan in Escherichia coli. The effects of nutritional shifts will be examined in wild type, and in mutants lacking specific regulatory functions. Global effects of the TyrR protein will be explored. in vitro band shift analyses will be performed with RNA polymerase, trp superrepressor, and various target operators, to determine the basis of the differential in vivo regulation of transcription initiation in the four operons controlled by the trp repressor of E. coli. in vivo experiments will be performed to determine the effects of replacing the normal repressor by a slowly dissociating superrepressor. Second and third site revertants will be isolated in tryptophan synthetase a mutants altered in catalytic residues; residue changes should reveal important features of the active site of this protein. The basis of the defects in Crawford's trpB repairable mutants will be examined. The amino and carboxy terminal segments of the tryptophan synthetase chains of E. coli will be systematically altered in an effort to explain the order of gene fusion that occured during evolution of the fused tryptophan synthetase structural gene of fungi. %%% Much of what is known about metabolism of the amino acid tryptophan in bacteria is due to the work of this laboratory. Although many details of the complex network of regulation controlling tryptophan synthesis and utlization can now be explained, the problem still offers many avenues of study that will provide information concerning a wide range of fundamental biochemical and genetic processes.

对大肠杆菌中调节色氨酸摄取、合成和利用的调节代谢网络的研究将继续进行。营养变化的影响将在野生型和缺乏特定调节功能的突变体中进行研究。我们将探讨TyrR蛋白的全局效应。体外带移分析将使用RNA聚合酶、trp超抑制因子和各种靶操作符进行，以确定由大肠杆菌trp抑制因子控制的四个操纵子在体内转录起始调控的差异基础。将进行体内实验，以确定用缓慢解离的超抑制因子取代正常抑制因子的效果。在催化残基改变的色氨酸合成酶突变体中分离出第二和第三位点的回复性；残基变化应揭示该蛋白活性位点的重要特征。研究Crawford trpB可修复突变体缺陷的基础。大肠杆菌色氨酸合成酶链的氨基末端和羧基末端将被系统地改变，以解释在真菌色氨酸合成酶结构基因融合进化过程中发生的基因融合顺序。我们对细菌中色氨酸代谢的了解，很大程度上要归功于这个实验室的工作。虽然现在可以解释控制色氨酸合成和利用的复杂调节网络的许多细节，但这个问题仍然提供了许多研究途径，将提供有关广泛的基础生化和遗传过程的信息。