A Single Amino Acid Substitution in Zinc Finger 2 of Adr1p Changes its Binding Specificity at two Positions in UAS1

Abstract

The two zinc fingers of the yeast transcription factor Adr1p recognize the 6 bp sequence TTG GAG site in which the first finger, a His-X 3-His finger, recognizes the G-rich triplet (GAG) and the second zinc finger, a His-X 4-His finger, recognizes the T-rich sequence (TTG). Mutations were introduced into the alpha-helical region of the second finger and the resultant mutant proteins were analyzed for DNA binding affinity and specificity in vitroand in vivo. Substitution His for Leu in the third position (+3) of the helix created a new binding specificity at two positions in the binding site. The mutant with His replacing Leu146(L146H) bound with high affinity to GGG GGG and with low affinity to TTG GGG. The single substitution at position +3 in the helix had the same effect on DNA binding specificity as substitution of the whole helix of the second finger with the helix of finger one. Changing Asp145 to Ala in the presence of His146 changed the apparent binding site of finger 2 to GT/CG. The L416H mutant zinc finger protein had the same binding specificity in vivoas in vitro. Changing the spacing between the His residues that ligand zinc in the second finger from four to three, the spacing found in the first finger of Adr1p and Zif268, did not alter the specificity or affinity of the wild-type or mutant protein. f2 f2 Abbreviations used: DMS, dimethylsulfate.