INVESTIGATION SF THE PYRIMIDINE PREFERENCE BY THE C-MYB DNA-BINDING DOMAIN AT THE INITIAL BASE OF THE CONSENSUS SEQUENCE

The principal determinant of the pyrimidine preference by the c-Myb DN A-binding domain at the initial base of the consensus sequence was inv estigated by mutation of both the protein and the DNA base pairs, with analysis by a filter binding assay. Amino acid residue 187 was reveal ed to interact with the pyrimidine base position, as estimated from ou r previous complex structure. Unexpectedly, since the pyrimidine prefe rence is retained even in the Gly(187) mutant, the principal origin of the base specificity should no occur via the direct-readout mechanism , but by an indirect-readout mechanism, namely in the intrinsic ''bend ability'' of the pyrimidine-purine step of the DNA duplex. A significa nt but rather small positive base pair roll is detectable in the confo rmation of DNA in complex with the C-Myb DNA-binding domain. Following the conventional chemical rules of the direct-readout mechanism, amin o acid mutagenesis at position 187 yielded several new base preference s for the protein.