BIOPHYSICAL INVESTIGATIONS ON THE MYB-DNA SYSTEM

The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs h ave been detected in the genes of proteins implying its role in a vari ety of regulatory functions. The protein has a DNA binding domain cons isting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variet y of investigations on the structure and interactions of the DNA bindi ng domain of Drosophila c-myb and its cognate DNA target sequences. Th e domain has been bacterially over-expressed by subcloning a segment o f the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has reve aled that the domain is largely helical in nature. Fluorescence invest igations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant pro tein is able to distinguish between specific and non-specific DNA targ ets in its binding and the interaction is largely electrostatic in nat ure in both cases. Dynamic fluorescence quenching experiments suggeste d that the DNA binding sites on the protein for specific and non-speci fic DNA targets are physically different. Most of the conserved trypto phans are associated with the specific DNA binding site. Simulated ann ealing and molecular dynamic simulations in a water matrix have been u sed to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues sho ws that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many resi due type and the types of residues involved in hydrophobic core format ion in the protein. In an attempt to see the DNA surface possibly invo lved in specific interaction with the protein, a three-dimensional str ucture of a 12 mer cognate DNA has been determined by NMR in conjuncti on with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction. (C) 1997 Elsevier Science B.V.