In vitro and in vivo studies of an Agrobacterium tumefaciens transcriptional factor BlcR

Transcription factors regulate gene expression by interacting with specific DNA promoter sequences and mediate the gene transcription initiation, thus play a critical role in many different important cellular processes. The activity of transcription factors can be regulated by post-translational modification and interactions with regulatory molecules, including small ligands which usually reflect environmental or physiological changes. The regulation of transcription factor activities by interactions with small ligands can be classified into three groups: the classical allosteric modulation, the assembly-mediated regulation, and the combination of both. This thesis focuses on molecular mechanism of a transcription factor BlcR from Agrobacterium tumefaciens whose regulation is mediated by its cognate effector ligand (succinate semialdehyde, SSA) via the combination mechanism. BlcR belongs to the i&barbelow;soc&barbelow;itrate l&barbelow;yase transcription r&barbelow;egulator (IclR) protein family that controls a wide range of cellular activities in response to environmental or growth variations. However, the biochemical and biophysical understanding of how IclR proteins function to control gene expression and how the interaction with the inducing ligands to modulate their activity are limited. BlcR is an experimentally amenable IclR-type protein for biochemical and biophysical characterizations, to reveal how the IclR-type proteins recognize their inducing ligands and target DNA sequences, how they interact with DNA, and how the effector ligands modulate their DNA binding affinity. In this thesis, we explored in vitro biochemical and structural and in vivo studies to understand the regulation mechanism of BlcR function by characterizing BlcR-DNA and BlcR-ligand interactions, as well as the effect of ligand on BlcR-DNA interaction and BlcR assembly. The outcome of this work will provide mechanistic understanding of the less-characterized yet biologically important IclR-type transcription factor family, and will contribute generally to our knowledge on the assembly-mediated regulation and the allosteric modulation of transcription factors.