Use of a ligand-screening procedure to study the interaction of S. cerevisiae alpha 2 repressor with its operator sequence.
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.
Lorimer
I A
IA
University of British Columbia.
Ho
C Y
CY
Smith
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005798:Genes, Bacterial / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D005809:Genes, Regulator / Q000502:physiology*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D009875:Operator Regions, Genetic / Q000502:physiology*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D009841:Oligonucleotides