Abstract
Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.
Highlights
Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils
We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex
Application of this model to mobility shift assays, for which the experimental observables are fractions of DNA free, of DNA bound and of DNA bound nonspecifically, yields the partition function given by Equation 1, the third term, which accounts for nonspecifically bound species, N is the size of the oligonucleotide in base pairs, n is the number of base pairs occluded by a nonspecifically bound IHF dimer, and j indexes species by stoichiometry of bound IHF
Summary
Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils It does so by binding in a sequence-independent manner throughout the genome. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. Indirect readout refers to recognition of aspects of DNA structure such as intrinsic curvature, topology of major and minor grooves, ordered water structures, local geometry of backbone phosphates, and flexibility or deformability Because both the local DNA structure and energy to deform DNA are themselves intrinsic sequence-dependent properties, the conserved sequences that distinguish binding sites necessarily include contributions from both direct and indirect mechanisms.
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