Designing zinc-finger ADR1 mutants with altered specificity of DNA binding to T in UAS1 sequences.

Yeast ADR1 contains two Cys2,His2 zinc fingers needed for DNA binding to the upstream activation sequence UAS1, with bases T5T6G7-G8A9G10 in the ADH2 promoter. Potential DNA-contacting amino acid residues at -1, +3, and +6 in the alpha-helical domains of ADR1's fingers one and two include RHR-RLR; however, the latter finger two residues Leu146 and Arg149 had not proved to be crucial for ADR1 binding, even though Leu146-T6 and Arg149-T5 interactions with UAS1 DNA were predicted. We altered Leu146 or Arg149 by PCR cassette mutagenesis, to study ADR1 mutant binding to 16 UAS1 variants of thymine bases T5 and T6. Mutation of Leu146 to His, making finger two (RLR) like finger one (RHR), decreased binding to wild type UAS1 having T6, but enhanced its binding strength to sequences having purines G6 or A6, similar to binding seen between finger one's His118 and base A9 of UAS1. Mutating Leu146 to Lys caused this finger two RKR mutant to bind strongly to both G6 and T6, possibly by lysine's amine H-bonding to the carbonyl of guanine or thymine. Specificity of ADR1 for UAS1 with T6 may thus be due to hydrophobic interaction between Leu146 and the T6 methyl group. ADR1 mutants with either His or Lys in the central +3 residue (146) of zinc finger two, which have Arg149 in the +6 alpha-helical position, bind with UAS1 mutant sequences having G5 very strongly, T5 strongly, A5 intermediately, and C5 weakly.(ABSTRACT TRUNCATED AT 250 WORDS)