Genetic selection for mutations that impair the co‐operative binding of lambda repressor

Abstract

Bacteriophage lambda repressor binds co-operatively to adjacent pairs of DNA target sites. A novel combination of positive genetic selections, involving two different operon fusions derived from P22 challenge phages, was used to isolate mutant lambda repressors that have lost the ability to bind co-operatively to tandem sites yet retain the ability to bind a strong, single site. These cb (co-operative binding) mutations result in 10 different amino acid changes, which define eight residues in the carboxyl-terminus of repressor. Because challenge phage derivatives may be applied to study essentially any specific protein-DNA interaction, analogous combinations of genetic selections may be used to explore the ways that a variety of proteins interact to assemble regulatory complexes.

Amino acid–amino acid contacts at the cooperativity interface of the bacteriophage λ and P22 repressors

“…We had suggested previously that D197 in cI and the corresponding D179 in P22c2 play essential roles in cooperativity on the basis of the fact that replacement of each of these aspartic acids with a glycine abolished cooperativity (Whipple et al 1994; see also Benson et al 1994). We hypothesized that the aspartic acid side chain might participate in an electrostatic bond at the dimer-dimer interface, and therefore, we sought a conserved basic residue as a candidate partner for D197.…”

“…Like cI, P22c2 has two domains, and the NTD mediates DNA binding whereas the CTD mediates dimerization and cooperativity (Poteete and Ptashne 1982). Both cI and P22c2 mutants that are specifically defective for cooperativity have been identified (Hochschild and Ptashne 1988;Valenzuela and Ptashne 1989;Beckett et al 1993;Benson et al 1994;Burz and Ackers 1994;Whipple et al 1994); most of these mutants, which bear amino acid substitutions in the CTD, manifest no defects in dimerization. Although cI and P22c2 exhibit considerable amino acid sequence homology in their CTDs (Sauer et al 1982), heterodimers do not form nor do the respective homodimers interact with one another.…”

“…The bacteriophage cI protein (the repressor, or cI) provides an example of cooperative binding to DNA that has been well characterized biochemically and genetically (for review, see Sauer et al 1990;Hochschild and Ptashne 1988;Beckett et al 1993;Benson et al 1994;Burz and Ackers 1994;Whipple et al 1994). cI, which functions as both a repressor and an activator of transcription, is a two-domain protein that binds each of its operator sites as a dimer (Ptashne 1992).…”

Dynamical perspective of protein-DNA interaction

“…λ-Repressor-operator sites interaction, particularly O R 1 and O R 2, is a key component of the λ-genetic switch and is a C-terminal domain is responsible for most protein-protein interactions (23); these interactions are essential for the co-operative binding (24,25) and for the functioning of the genetic switch (11,26). We will discuss the conformational change of the repressor protein and the key dynamical timescales involved in such specific protein-DNA interactions (28).…”

“…The N-terminal domain interacts with the DNA (16), while the C-terminal domain is responsible for most protein-protein interactions (23); these interactions are essential for the co-operative binding (24,25) and for the functioning of the genetic switch (11,26).…”