REGULATION OF ISOLEUCINE/VALINE BIOSYNTHESIS

异亮氨酸/缬氨酸生物合成的调节

• 批准号: 3275283
• 负责人: ROBERT P LAWTHER
• 金额: $ 9.91万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1986-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275283
• 关键词: Escherichia coli k12; aminoacid biosynthesis; gene expression; genetic manipulation; genetic mapping; genetic transcription; isoleucine; microorganism metabolism; molecular cloning; mutant; nucleic acid sequence; operon; valine; virus DNA

We are interested in the mechanism by which a cell coordinates the expression of the various genes required for its growth. The genes for isoleucine/valine biosynthesis are separated into four separate transcriptional units. That are regulated independently but respond to similar signals (i.e., intracellular concentrations of leucine, isoleucine and valine). It is important for optimal cell growth that the level of the products of these genes be coordinated to supply the required balance of these amino acids. Four of the genes combine to form the ilvGEDA operon, which contains multiple regulatory sites so as to achieve the requisite balance of gene products. The primary site of regulation is the promoterattenuator proximal to ilvG. Secondary sites within the operon include: two internal promoters and an internal termination site. Presently, we have only a rudimentary understanding of how these elements combine to control the level of the enzymes encoded by these genes. In order to study the interrelationship of the regulatory components of the ilvGEDA operon, we propose to examine the role of each regulatory element by using gene fusions constructed in vitro by recombinant DNA techniques. In each fusion an ilv regulatory element will be inserted into a plasmid such that expression of a plasmid gene is dependent on the inserted ilv DNA. These plasmids will then be subjected to in vitro mutagenesis and the altered plasmids examined for altered gene expression. Subsequent analysis by in vitro transcription and DNA sequence determination will facilitate our establishing the nature and character of each regulatory element. Because the coordination of the expression of sets of genes is essential for cellular function, we believe it is important to determine the mechanisms by which this coordination is achieved. The genes for isoleucine/valine biosynthesis form such a set. Because the DNA of these genes has been isolated and characterized, they are readily accessible for detailed analysis of their regulation.

我们感兴趣的是细胞协调细胞的机制。 其生长所需的各种基因的表达。 的基因 异亮氨酸/缬氨酸生物合成分为四个独立的 转录单位 它们是独立调节的，但对 类似的信号（即，细胞内亮氨酸、异亮氨酸浓度 和缬氨酸）。 对于最佳细胞生长来说，重要的是， 这些基因的产物被协调，以提供所需的平衡， 这些氨基酸。 四个基因联合收割机形成ilvGEDA操纵子， 其包含多个调节位点，以实现所需的 基因产物的平衡。 调节的主要场所是 在ilvG近端的启动子衰减子。 操纵子内的次级位点 包括：两个内部启动子和一个内部终止位点。 目前，我们对这些元素如何 联合收割机来控制这些基因编码的酶的水平。 在 为了研究的监管组成部分的相互关系， ilvGEDA操纵子，我们建议检查每个调控元件的作用 通过使用重组DNA技术体外构建的基因融合体。 在每个融合体中，将ILV调节元件插入质粒中 使得质粒基因的表达依赖于插入的ILV DNA. 然后将这些质粒进行体外诱变， 检查改变的质粒以改变基因表达。 后续分析 通过体外转录和DNA序列测定将有助于 我们确定每个监管要素的性质和特征。 因为基因表达的协调是至关重要的 对于细胞功能，我们认为重要的是确定 实现这种协调的机制。 的基因 异亮氨酸/缬氨酸生物合成形成这样一组。 因为它们的DNA 基因已被分离和表征，它们很容易获得， 详细分析其监管。