Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers.
The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity.
Darling
P J
PJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Holt
J M
JM
Ackers
G K
GK
D000495:Allosteric Site; D010582:Bacteriophage lambda / Q000737:chemistry*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D009154:Mutation; D009875:Operator Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013698:Templates, Genetic; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D003850:Deoxyribonuclease I