Site-specific enthalpic regulation of DNA transcription at bacteriophage lambda OR.
Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration. The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions. It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions. By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small. This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry 30, 7817-7821] indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization. The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems. However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Koblan
K S
KS
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Ackers
G K
GK
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D013696:Temperature; D013816:Thermodynamics; D013950:Thymus Gland / Q000737:chemistry; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA