This project will develop a new method to map DNA-protein interactions using 15N NMR in combination with specific labeling. The Cro protein-operator system of bacteriophage Lambda will be used to establish the method. Cro is the smallest repressor molecule of known structure. The affinity of Cro for a specific operator nucleotide sequence has been documented by chemical and nuclease protection experiments. Double stranded oligomers of DNA will be prepared from 15N labeled nucleotides. NMR resonances of 15N are very sensitive to hydrogen bonding. Thus, when the protein binds to the DNA with formation of H-bonds between the Nitrogen atoms of the bases and the amino acid side chains, the resonance from individual nitrogen atoms will undergo shifts. In this way, the specific atoms of the bases in the DNA which are interacting with the protein will be mapped. After mapping the H-bonds of the Cro-operator complex, the method will be applied to the Lambda Mnt- protein which has significant sequence homology to the Cro protein Once the method has been established and tested on Cro and Mnt it will be ready to be used to investigate eukaryotic regulation systems and to develop a database correlating 15N NMR shifts with various types of DNA-protein interactions.
