Molecular Cloning, Optimization of Expression and Functional Characterization of the Global Transcriptional Regulator...

In-vivo microarray analysis on murine model of Tuberculosis had identified a novel transcriptional regulator MosR, which regulates the expression of ~ 163 genes, many of which are important for intracellular persistence of dormant Mycobacterium tuberculosis. MosR therefore, may be considered as potential target for anti-tuberculosis drug discovery. In this work, this important regulator has been cloned, expressed, purified to homogeneity, and characterized to some extent. MosR is found to cooperatively bind within the ORF of the previous gene rv0347. The environmental conditions for most favorable interaction between MosR and its cognate DNA are determined to be 8.0 pH, 25 mM NaCl, and 25 ℃ temperature. The equilibrium dissociation constant (K_d) for MosR-DNA interaction is determined to be 0.23 ± 0.02 µM under the optimized conditions. MosR is composed of mostly α-helices with minor β-sheets and β-turns as major secondary structural elements. Interestingly, MosR is found to harbor a highly conserved homeodomain and a long N-terminal arm which might be involved in binding to specific DNA, supported by the predicted three-dimensional structure. The tertiary structure of MosR is found to be stable under different pH and salt concentrations while secondary structure undergoes distinct conformational changes. MosR is unable to maintain its structure and DNA-binding activity at a temperature more than 35 ℃ and forms microaggregate in solution which suggests that it is a moderately thermosensitive protein. These information would be useful for in-vitro screening and validations of inhibitors against MosR in a high throughput manner.