Use of a ligand-screening procedure to study the interaction of S. cerevisiae alpha 2 repressor with its operator...

A simple and rapid screening procedure was developed to study the interaction of the S. cerevisiae alpha 2 repressor with its operator sequence. An E. coli expression vector was constructed in which the alpha 2 coding sequence was placed under control of the lac promoter. Bacterial colonies containing this vector could be lysed and assayed directly for binding of wild-type and mutant operator sequences when grown on nitrocellulose filters. alpha 2 assayed in this way showed the same sequence specificity as determined in vivo. Pools of mutant alpha 2 repressors in which the codons for Arg185 or Ser181 in the homeodomain region were randomized were created by cassette mutagenesis. These pools of mutants were screened with the wild-type operator sequence to determine allowed amino acid substitutions at each position. Results suggest that both Arg185 and Ser181 have a role in high affinity operator binding.