Since the lac operon has been the paradigm for gene regulatory systems, our efforts have been focused on obtaining structural information on the repressor operator complex. To this end, a series of DNA binding domain fragments and variants have been cloned and expressed for solution multinuclear multidimensional NMR analysis, both as proteins and protein DNA complexes. In collaboration with M. Lewis of the Department of Biochemistry and Biophysics, the three-dimensional structures of (1) lac repressor alone, (2) lac repressor with inducer, and (3) complexes of intact tetrametic lac repressor, and operator DNA have been solved.
An interesting result from our group is the solution structure of A-tract DNA. This variation from the average B form has been studied for two decades. Several X-ray structures by other groups have been not consistent with the body of solution properties of this family of DNA sequences. Our NMR solution structure shows that the bend In the helix axis is actually 90 degrees away from the bend plane in the crystal structures. The variation of nucleic acid structures as a function of sequence and solvent conditions is an important consideration. In related experiments, we have exploited the use of fluorescence depolarization of bound ethidium bromide to investigate hydrodynamic size and shape of RNA structures, e.g., tetraloops and ribozymes. These issues have also become important as we are finding variations in mRNA accessibility to antisense probes.
In collaboration with the late Alan Gewirtz we demonstrated that anti-sense oligonucleotides actually find their complementary sequences in the cell.