COOPERATIVE NONSPECIFIC DNA-BINDING BY OCTAMERIZING LAMBDA-CI REPRESSORS - A SITE-SPECIFIC THERMODYNAMIC ANALYSIS

Relationships between dimerization and site-specific binding have been characterized previously for wild-type and mutant cI repressors at th e right operator (O-R) of bacteriophage lambda DNA. However, the roles of higher-order oligomers (tetramers and octamers) that are also form ed from these cI molecules have remained elusive. In this study, a cle ar correlation has been established between repressor oligomerization and nonspecific DNA-binding activity. A modification of the quantitati ve DNase I footprint titration technique has been used to evaluate the degree of saturation of non-specific, O-R-flanking lambda DNA by cI r epressor oligomers. With the exception of one mutant, only those repre ssors capable of octamerizing were found to exhibit non-specific DNA-b inding activity. The non-specific interaction was accurately modeled u sing either a one-dimensional, univalent, site-specific Ising lattice approximation, or a more traditional, multivalent lattice approach. It was found that nonspecific DNA-binding by repressor oligomers is high ly cooperative and energetically independent from site-specific bindin g at O-R. Furthermore, the coupling free energy resolved for non-speci fic binding was similar to that of site-specific binding for each repr essor, suggesting that similar structural elements may mediate the coo perative component of both binding processes. It is proposed that the state of assembly of the repressor molecule modulates its relative aff inity for specific and non-specific DNA sequences. These specificities are allosterically regulated by the transmission of assembly-state in formation from the C-terminal domain, which mediates self-association and cooperativity, to the N-terminal domain, which primarily mediates DNA-binding. While dimers have a high affinity for their cognate sites within ORI tetramers and octamers may preferentially recognize non-sp ecific DNA sequences. The concepts and findings developed in this stud y may facilitate quantitative characterization of the relationships be tween specific, and non-specific binding in other systems that utilize multiple modes of DNA-binding cooperativity. (C) 1998 Academic Press.