Energetics of DNA Recognition by the bZIP Transcription Factors

Johns Hopkins University, Baltimore MD

Investigators

Abstract

The aim of this project is to determine the energetic basis of the structure of bZIP transcription factors and their specific complexes with DNA. The bZIPs form one of the largest families of transcription factors regulating gene activity in eukaryotes. Their study by physical methods is of particular interest for several reasons: (a) they all have the same overall structure and operate as dimers, varying subtly both in the dimerization domain (the leucine zipper) and in the DNA recognition domains (the basic segments), thus providing immense variability both in DNA recognition and induced structural change, all of which is important for transcriptional activation/repression; (b) their DNA recognition domains are unfolded in the absence of DNA but fold upon binding their cognate sequence; and (c) bZIP binding results in some distortion of the DNA. Notwithstanding numerous publications dedicated to the bZIP transcription factors, not much is known about the energetic bases of formation of these homo- and hetero-dimers and their interaction with DNA. This is because of a number of methodological problems, which are faced in studying the interactions of dimeric, and partly unfolded proteins with DNA. Their solution requires a combined thermodynamic investigation of the assembly of these dimeric molecules from their separated chains and the formation of their complexes with different binding sites at various temperatures and salt concentrations by microcalorimetric (DSC and ITC) and optical methods (CD, fluorescence anisotropy and FRET). Such a complex approach permits one to specify quantitatively, in thermodynamic terms the electrostatic and non-electrostatic forces involved in formation of these complexes: i.e. in the Gibbs energy, the enthalpy and the entropy. This opens prospects for modeling the mechanism of formation of these complexes based on the existing structural information. The methodology of this combined approach for such complicated systems was developed in the PI's group. Preliminary studies of one representative of the bZIP family, GCN4, have shown its effectiveness. Systematic studies of other representatives of this family of transcription factors will show how general these features are and will clarify their functional role, particularly of incorporated water in recognition of the specific binding sites. The planned studies of a set of closely related bZIPs will further develop the general methodology for studying the mechanisms of assembly of multi-molecular complexes, which is increasingly attracting attention. These studies will also significantly impact the training of young scientists in using advanced microcalorimetric and optical methods in the study of the mechanism of formation of specific protein-DNA complexes. Toward this end, the PI has developed courses that combine a traditional lecture format with the laboratory experience in which the students get to learn the most advanced microcalorimetric instruments.