Molecular and structural characterization of global transition state regulators AbrB and Abh from Bacillus subtilis.

摘要

Bacteria remarkably and constantly adapt to their surrounding environment, especially in times of considerable environmental stress. These responses range from secretion of toxins, antibiotics and complete physiological transformations leading to the development of highly resilient spores resistant to heat, sunlight, chemicals and drugs. Due to the constant flux in environmental conditions, bacteria spends most of its life in this critical period, the transition-state. Proteins involved in the activation of genes required for survival are called transition-state regulators, a novel family of DNA binding proteins. Two defining characteristics describe transition-state regulator proteins: (1) ability to recognize a multitude of genes with no consensus sequence and (2) sole decision making policy of the cell is controlled by this select group proteins to monitor and regulate hundreds of cellular pathways. Despite their vital and increasingly common role, there is a paucity of information. Bacillus subtilis and B. anthracis both possess transition-state regulators responsible for gene expression during transition from vegetative to post-exponential growth. This dissertation focuses on investigating the protein-DNA interactions of the transition state regulators, antibiotic resistance protein B (AbrB) and antibiotic resistance protein h (Abh) from B. subtilis and B. anthracis to develop a detailed general model describing recognition and interaction mechanisms. Biophysical characterization of AbrBN and AbhN show that multimerization plays an essential role in binding to DNA targets. High-resolution NMR structure analysis was carried out to refine the structure of AbrBN and to solve the structure of AbhN, both showing dimeric topologies. These studies employed a complementary mutagenesis, ESI-MS, NMR, as well as a multiple spectroscopic technique approach. The study concluded with a respectable model of AbrB's, and transition state regulators in general, interaction with DNA. The unique ability to bind over 60 genes with no consensus sequence relies on conformational flexibility of both protein and DNA. Furthermore, the solution structure of the N-terminal domain of Abh provided the stature needed to declare the transition-state regulator family a unique and characterized DNA binding family.