The objective of this work is to further our understanding of the molecular components and mechanisms involved in the regulation of gene expression. This proposal is based on our studies which have lead to the identification of the transcriptional control elements that regulate inducible tetracycline resistance in Escherichia coli K12. We have localized the gene for a specific repressor function and determined the DNA sequence of a region containing multiple promoter-operator sites for the resistance genes. Our objectives now are to: 1) examine in greater detail the primary structure and function of these regulatory elements, and 2) define the overall gene structure and organization of the resistance determinant. We will test a model, consistent with our current data, that transcription of the repressor gene and transcription of the resistance gene(s) are initiated at divergent and functionally overlapping promoter-operator sites, and that the repressor gene is subject to negative autogenous regulation. We will make extensive use of gene fusions between the regulatory elements of the resistance genes and the lacZ structural gene in order to isolate and characterize regulatory mutations. DNA and RNA sequencing, recombinant DNA, and in vitro transcription methodologies will be employed in these investigations.
