Isolation of lambda repressor mutants with defects in cooperative operator binding.
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.
Beckett
D
D
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
Burz
D S
DS
Ackers
G K
GK
Sauer
R T
RT
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins; D004734:Energy Metabolism; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D015500:Chloramphenicol O-Acetyltransferase