The objective of this project is to study the structure and dynamics of regulatory DNA sequence complexes with their repressor or activators. We will approach this using three systems: a) The operator of the lac operon; b) the two sets of lambda phage operators OR and OL. c) The lambda phage sites of action of CII protein, PL and PE. The lac system represents the regulation of gene expression in response to environmental change. The lambda phage operators are an example of a developmental, regulatory switch. The lambda phage CII system is a positive transcriptional regulatory system necessary for the activation of gene products leading to the establishment of the lysogenic state. We will make extensive use of nuclear magnetic resonance (NMR) spectroscopy to study these DNA target sequences and their contacts with the regulatory proteins. The NMR observations will take advantage of variations of the regulatory DNA sequences made by synthetic methods. Using operator or promoter DNA sequences with specific nuclear spin labels we will study the changes in the DNA structure and the nature of the contracts that lead to the specificity of these proteins. Preliminary data indicate the possibility of sequence dependent local heterogeneity in structure and dynamics of DNA regulatory regions. It is proposed that the above program will show the way to approach parallel studies with eukaryotic enhancers and other regulatory elements.
