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Isolation of lambda repressor mutants with defects in cooperative operator binding
D Beckett1, D S Burz, G K Ackers
1Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
Abstract:
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.
Insights
Researchers identified a specific mutation in the lambda repressor protein that disrupts cooperative DNA binding. This finding is crucial for understanding transcriptional regulation and developing new tools for genetic studies.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The lambda repressor protein controls gene expression in bacteriophages.
- Cooperative binding of repressor dimers to operator sites is essential for efficient gene regulation.
- Understanding the molecular basis of cooperativity is key to deciphering gene circuit logic.
Purpose of the Study:
- To design a novel screening system for identifying lambda repressor mutants with defects in cooperative binding.
- To isolate and characterize single amino acid substitutions affecting repressor cooperativity.
- To elucidate the role of specific residues in mediating protein-protein interactions during DNA binding.
Main Methods:
- Construction of a hybrid operator-promoter DNA region for screening.
- Isolation and purification of mutant lambda repressor proteins.
- DNA binding assays to measure operator site affinity.
- Quantitative footprinting to determine interaction free energy between bound dimers.
Main Results:
- A screening system successfully identified lambda repressor mutants with impaired cooperativity.
- The Glycine147 to Aspartic acid (GD147) mutation was isolated, affecting the C-terminal domain.
- The GD147 mutant exhibits normal binding to single operator sites but defective cooperative binding.
- Quantitative footprinting revealed a complete loss of interaction energy between repressor dimers at adjacent operators (OR1 and OR2) for the GD147 mutant.
Conclusions:
- The Gly147 residue is critical for mediating cooperative interactions between lambda repressor dimers.
- The developed screening system is effective for studying protein-protein interactions in DNA binding.
- This work provides insights into the structural determinants of cooperativity in transcriptional repressors.