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 five transcriptional units. A variety of molecular signals act as effectors of the expression of the ilv genes. These include both ilv specific regulators (such as the amino acid end products and proteins) and general or global controls (such as ppGpp and protein factors). These molecular species affect ilv gene expression by different mechanisms (ie, transcription initiation vs. attenuation). The number and spectrum of effectors reflects that isoleucine, leucine, and valine constitute an above average percentage of the amino acids present in protein. Thus, their synthesis represents a major commitment of cellular resources and it is essential the cell be able to optimally modulate the expression of these genes in response to alternative physiological conditions. Five the genes combine to form the ilvGMEDA operon, which contains multiple regulatory sites so as to achieve the requisite balance of its gene products. The primary site of regulation is the promoter-attenuator proximal to ilvG. Secondary sites within the operon include: two internal promoters and internal termination sites. The trans effectors (ppGpp and proteins) appear to regulate expression at the 5' promoter. In order to study the factors involved in the regulation of the operon, we will employ the ilv-galK fusions which we have constructed. These plasmids will be studied using a variety of techniques. This will include: (1) oligonucleotide mutagenesis; (2) in vivo analysis of proein interactions; and (3) in vitro analysis of RNAP interactions. Also, we have defined a site upstream of the promoter, which is required for maximal expression from the ilv promoter and propose to isolate the protein(s) that act at this site. 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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