Linked Equilibria in Biotin Repressor Function: Thermodynamic, Structural, and Kinetic Analysis

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Publisher Summary Regulation of transcription initiation involves assembly of multisubunit protein complexes at the transcription control regions of genes. To ensure that transcription initiation occurs in response to appropriate cellular conditions and with proper timing the assembly of these regulatory complexes is subject to control. Thus, many transcription regulators are allosteric proteins. As such their affinities for specific DNA sites are modulated either by small ligand binding or posttranslational modification. The focus of this chapter is on methods to examine the coupled reactions in the biotin repressor. Before describing developments in examining linked equilibria in the biotin repressor, some of the basic features of the system are reviewed. A model of the three-dimensional structure of the unliganded or aporepressor is shown here. In this state, the protein is monomeric and is composed of three domains: the N-terminal DNA-binding domain, the central domain, and the C-terminal domain. In addition to its structured core, the central domain is characterized by four surface loops that are partially disordered in the aporepressor. Binding of biotin and bio-5’-AMP to the biotin repressor (BirA) are characterized by equilibrium constants of approximately 4 ×10 -8 and 4×10 -11 M, respectively, in the standard buffer conditions utilized in laboratory. Furthermore, kinetic analysis of both binding processes by stopped-flow fluorescence reveals that binding is a two-step process, involving initial formation of a collision complex followed by a conformational change in the protein. This chapter shows that combined structural, thermodynamic, and kinetic analysis of the biotin repressor has proved powerful in obtaining information about the linked equilibria in this system. This linkage is the key to the regulation of function in the complex biotin regulatory system. Results of qualitative analysis of many biological regulatory processes indicate that multiple linkages are common in modulating the function of participating macromolecules. Application of the approaches utilized in studies of the biotin repressor system to these other systems will yield information that will raise the understanding of the biology to a quantitative level.