Regulation of Amino Acid Biosynthesis

天冬氨酸激酶在细菌氨基酸生物合成中起着核心作用，其活性通过复杂的控制机制来调节细菌的生理需要。枯草芽孢杆菌有两种天冬氨酸激酶，其中一种（天冬氨酸激酶I）受细胞壁成分二氨基乳酸调节，而另一种（天冬氨酸激酶II）是本研究的重点，由赖氨酸控制，不同寻常的是由两种不同类型的亚基组成，这两种亚基是由同期重叠基因独立翻译的。这些和它们的控制区已被克隆，它们的核苷酸序列已被确定。本课题旨在将序列信息应用于天冬氨酸激酶合成控制机制的研究以及该酶本身结构与功能之间的关系。拟议的研究将使用寡核苷酸定向诱变来改变控制区的特定位点以及编码蛋白质结构的基因，期望对这些结构扰动的研究将揭示控制和功能背后的机制。另一个目的是对天冬氨酸激酶II基因位点周围的基因进行测序，并阐明它们的调节和代谢关系。本研究研究的枯草芽孢杆菌有两种生长模式：正常环境条件下的营养生长模式和饥饿或其他应激条件下的产孢生长模式。后一个过程导致形成高度持久的孢子，这些孢子可以分散，并随后在条件允许营养生长的地方发芽。营养性生长的细菌在接受特定的刺激开始产孢后，必须改变其整个内部生理机能以满足新的生长模式的要求。某些关键酶在两种不同的生长模式下起着不同的生理作用，因此受到一系列非常复杂的控制，使细菌从营养生长过渡到产孢。这是一项阐明这些控制机制的建议；它可能揭示了以前未知的基因表达控制机制。芽孢杆菌细胞从营养生长到产孢的转变是一个发育开关，可以作为高等生物胚胎组织分化的概念模型。因此，本建议中的工作可能间接说明后一种进程。

The enzyme aspartokinase plays a central role in bacterial amino acid biosynthesis, and its activity is attuned to the physiological needs of the bacterium by complex control mechanisms. Bacillus subtilis has two aspartokinases, one of which (aspartokinase I) is regulated by the cell wall constituent diaminopimelate, while the other (aspartokinase II), the major focus of this research, is controlled by lysine and is unusual in being composed of two different types of subunits which are independently translated from in-phase overlapping genes. These and their control regions have been cloned and their nucleotide sequence has been determined. This proposal aims to apply the sequence information to the study of the mechanisms that control aspartokinase synthesis and of the relationship between structure and function of the enzyme itself. The proposed research will use oligonucleotide directed mutagenesis to alter specific sites in the control regions as well as in the genes encoding the protein structure, in the expectation that the study of these structural perturbations will reveal the mechanisms that underlie control and function. Another aim is the sequencing of the genes that surround the genetic locus of aspartokinase II and the elucidation of their regulatory and metabolic relationship. Bacillus subtilis, the species of bacterium under study in this proposal, has two modes of growth, vegetative under normal environmental conditions, and sporulation under conditions of starvation or other types of stress. The latter process results in the formation of highly durable spores which can be dispersed, and can subsequently germinate when and where conditions permit vegetative growth. Vegetatively growing bacteria, upon receiving specific stimuli to begin sporulation, must alter their entire internal physiology to meet the requirements of the new mode of growth. Certain key enzymes, which play different physiological roles under the two different modes of growth, are thus under a very complex series of controls, which permit the transition of the bacterium from vegetative growth to sporulation. This is a proposal to elucidate the mechanisms of these controls; it could possibly reveal previously unknown mechanisms of control of gene expression. The transition of Bacillus cells from vegetative growth to sporulation is a developmental switch which can serve as a conceptual model for the differentiation of embryonic tissues in higher organisms. The work in this proposal may thus shed indirect light on the latter process.