DESIGNING ZINC-FINGER ADR1 MUTANTS WITH ALTERED SPECIFICITY OF DNA-BINDING TO T IN UAS1 SEQUENCES

Yeast ADR1 contains two Cys(2),His(2) zinc fingers needed for DNA bind ing to the upstream activation sequence UAS1, with bases T(5)T(6)G(7)- G(8)A(9)G(10) in the ADH2 promoter. Potential DNA-contacting amino aci d residues at -1, +3, and +6 in the alpha-helical domains of ADR1's fi ngers one and two include RHR-RLR; however, the latter finger two resi dues Leu146 and Arg149 had not proved to be crucial for ADR1 binding, even though Leu146-T-6 and Arg149-T-5 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 T-5 an d T-6. Mutation of Leu146 to His, making finger two (RLR) like finger one (RHR), decreased binding to wild type UAS1 having T-6, but enhance d its binding strength to sequences having purines G(6) or A(6), simil ar to binding seen between finger one's His118 and base A(9) of UAS1. Mutating Leu146 to Lys caused this finger two RKR mutant to bind stron gly to both G(6) and T-6, possibly by lysine's amine H-bonding to the carbonyl of guanine or thymine. Specificity of ADR1 for UAS1 with T-6 may thus be due to hydrophobic interaction between Leu146 and the T-6 methyl group. ADR1 mutants with either His or Lys in the central +3 re sidue (146) of zinc finger two, which have Arg149 in the +6 alpha-heli cal position, bind with UAS1 mutant sequences having G(5) very strongl y, T-5 strongly, A(5) intermediately, and C-5 weakly. Mutation of Arg1 49 to Gln decreased DNA binding to G(5) and T-5 while increasing its b inding to A(5) and C-5 probes, thus showing the expected Arg149 intera ction with G(5) or T-5 in UAS1 DNA.