MOLECULAR BASIS OF CONTROL IN THE L-ARABINOSE OPERON

描述了遗传和生化实验，这些实验将分析 控制L-阿拉伯糖操纵子表达的分子相互作用 大肠埃希菌。AraBAD操纵子和Arac调控基因启动子 将被使用体外诱变的突变体饱和。这些方法 将使用的包括(1)合成寡核苷酸定向突变，(2) 羟胺的重度诱变；(3)定点化学 修改。在体外产生的突变将转移到 染色体，分析它们对ara基因表达的影响。 一个假定的多肽可能的功能意义 将对Arac LeadermRNA进行评估。将在DNA上进行比较 Ara启动子和其他来源的arc基因的序列水平 肠杆菌科。小分子与纯化的ArAc的相互作用 调节蛋白将用荧光分光光度法进行研究。 最后，我们将尝试确定三维结构 有无L-阿拉伯糖和Arac对Arac调节蛋白的影响 与Arai和Arao DNA络合的蛋白质。

Genetic and biochemical experiments are described which will analyze the molecular interactions that control expression of the L-arabinose operon in Escherichia coli. The araBAD operon and araC regulatory gene promoters will be saturated with mutants using in vitro mutagenesis. The methods used will include (1) synthetic oligonucleotide-directed mutagenesis, (2) heavy mutagenesis with hydroxylamine, and (3) site-directed chemical modification. Mutations created in vitro will be transferred to the chromosome for analysis of their effect on expression of the ara genes. The possible functional significance of a putative polypeptide coded for by araC leader mRNA will be assessed. A comparison will be made at the DNA sequence level of ara promoters and the araC gene from other Enterobacteriaceae. The interaction of small molecules with purified araC regulatory protein will be studied by spectrofluorimetric methods. Finally, an attempt will be made to determine the 3-dimensional structure of the araC regulatory protein with and without L-arabinose and araC protein complexed with araI and araO DNA.