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.
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