Proton-linked contributions to site-specific interactions of lambda cI repressor and OR.
The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.
Senear
D F
DF
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Ackers
G K
GK
D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D009710:Nucleotide Mapping; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D012097:Repressor Proteins / Q000378:metabolism* / Q000648:ultrastructure; D014157:Transcription Factors / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism* / Q000648:ultrastructure; D054334:Viral Regulatory and Accessory Proteins
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011522:Protons; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D003850:Deoxyribonuclease I