Isolation of .lambda. repressor mutants with defects in cooperative operator binding

• Published in: Biochemistry (Easton) Vol. 32; no. 35; pp. 9073 - 9079
• Main Authors: Beckett, Dorothy, Burz, David S, Ackers, Gary K, Sauer, Robert T
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 07-09-1993
• Subjects: Base Sequence; Biological and medical sciences; Chloramphenicol O-Acetyltransferase - genetics; Chloramphenicol O-Acetyltransferase - isolation & purification; DNA-Binding Proteins; Energy Metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; Operator Regions, Genetic - genetics; phage lambda; Promoter Regions, Genetic - genetics; Recombinant Fusion Proteins - isolation & purification; Repressor Proteins - genetics; Repressor Proteins - isolation & purification; Repressor Proteins - metabolism; Sequence Analysis, DNA; Transcription. Transcription factor. Splicing. Rna processing; Transformation, Genetic; Viral Proteins; Viral Regulatory and Accessory Proteins; Virus; Molecular cooperativity; Transcription operator; Siphoviridae; Site directed mutagenesis; Molecular interaction; Transcription repressor; Phage lambda; In vitro; Structure function relationship; Phage
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00086a012

A hybrid operator-promoter region was designed to aid in a screen for cooperativity mutants of the lambda repressor. In this system, lambda repressor mutants with defects in pairwise cooperative binding are unable to act as efficient transcriptional repressors. Four single amino acid substitutions in the C-terminal domain of the repressor were isolated. Studies of the DNA binding properties of the purified mutant proteins show that a repressor bearing the Gly147-->Asp mutation binds with normal affinity to single operator sites but is defective in pairwise cooperative site binding. Quantitative footprinting studies show that the free energy of interaction between repressor dimers bound at operator sites OR1 and OR2 is reduced from -2.4 kcal/mol for the wild-type repressor to 0 kcal/mol for the GD147 mutant.