Genetic and Biochemical Studies of the Tryptophan Operon

对调节大肠杆菌中色氨酸的吸收、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检验。本课程将探讨TyrR蛋白的全球效应。我们将用RNA聚合酶、Trp超抑制子和各种靶向操纵子进行体外带移分析，以确定在体内对由E.Coli的Trp抑制子控制的四个操纵子转录起始的不同调控的基础。将进行体内实验，以确定用缓慢解离的超级抑制物取代正常抑制物的效果。在色氨酸合成酶中将分离出第二和第三位点回复突变，这是一种在催化残基中发生改变的突变；残基的变化将揭示该蛋白质活性部位的重要特征。将研究Crawford的trpB可修复突变体中缺陷的基础。为了解释真菌色氨酸合成酶结构基因进化过程中基因融合的顺序，将系统地改变大肠杆菌色氨酸合成酶链氨基和羧基末端片段。已知的有关细菌中氨基酸色氨酸新陈代谢的大部分是由于这个实验室的工作。尽管现在可以解释控制色氨酸合成和利用的复杂调控网络的许多细节，但这个问题仍然提供了许多研究途径，将提供关于广泛的基本生化和遗传过程的信息。

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.