X Phage cro Repressor Interaction with DNA

We show here the classification of the 'H NMR spectrum of the cro repressor using the combined approach of selective deuteration of the tyrosine aromatic rings, nuclear Overhauser enhancement effects (Wagner and Wuthrich, 1979), and twodimensional spin echo correlated spectroscopy (Nagayama and Wiithrich, 1981). The two-dimensional SECSY' spectrum allows the determination of which resonances are related by through bond J connectivities. Thus, all the resonances belonging to each given amino acid type can in principle be identified. The data here classify and identify the aromatic resonances in the NMR spectra. Our results show that the interpretation of the 'H NMR spectrum of cro repressor presented by Iwahashi et al. (1982) using only one-dimensional NMR is incorrect. We present an examination of the aromatic residues of the cro repressor using 'H and "F NMR spectroscopy. By using a plasmid which has the cro structural gene under control of the lac operator-promotor (Roberts et al., 1979), it is possible to obtain large amounts of cro repressor for NMR spectroscopy. Also, the biosynthetic incorporation of 3-fluorotyrosine and 3-fluorophenylalanine into the cro repressor is possible using the same plasmid since the cro repressor expression is under control of the lac operator and promoter. By transforming the Escherichia coli cells containing the lac repressor with this plasmid, the cro gene is repressed until a lac operon inducer is added to the culture. The induction of cro repressor synthesis, with the simultaneous addition of 3-fluorotyrosine, phenylalanine, and tryptophan, results in the incorporation of 3-fluorotyrosine into the cro repressor. This is essentially the same procedure we have used for the incorporation of 3fluorotyrosine into the lac repressor where induction was