Altered Cro repressors from engineered mutagenesis of a synthetic cro gene.

Abstract

A portion of the gene coding for the Cro repressor protein of bacteriophage .lambda. was chemically synthesized, incorporating base pair changes that generate restriction endonuclease site without altering the amino acid coding sequence. These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations. Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding .lambda. operator OR3 in vivo. Mutations directed into the region of the cro gene encoding the .alpha.-3 helix produced altered Cro proteins with a range of affinities for operator DNA. These changes suggest which amino acids play an important role in Cro-OR3 complex formation.