The Identification Of Key Amino Acid Residues And Recognized Bases Of A Global Regulator Ms6564

Ms6564, a member of TetR family transcriptional regulator, regulates a large number of gene expression in Mycobacterium smegmatis. Although Ms6564 positively regulates the expression of cell cycle and DNA damage/repair genes as an activator, and negatively affects mycobacterial cell growth and gene mutation rates, the molecular mechanism between the regultor and DNA is unclear. Accordingly to a recent crystal structure of Ms6564-DNA complex, we further studied on the molecular basis of the interaction between Ms6564 and DNA as follows:(1)10 point mutated and 4 N-domain deleted proteins of Ms6564 were designed and successfully purified. The 47 lysine (Lys) of Ms6564 was characterized as a key amino acid residues essential for the DNA-binding ability of Ms6564 by electrophoretic mobility shift assay.(2)4 mutant DNA fragments were designed and synthesized. Then a new conserved motif containing GXCXXTXCGXCXXGXC core was characterized as the recognition sequence for Ms6564 by electrophoretic mobility shift assay.(3)Using new characterized motif to search the promoter regions of M. smegmatis genomic sequence, a total of 143 target promoters was found to be probably regulated by Ms6564. In which,34 promoters were shown to be bound by Ms6564 using ChIP assay in vivo. In addition, we confirmed that Ms6564 actually regulated expression of these genes by qRT-PCR assay.The current study has identified a new conserved motif for the recognition of Ms6564 and its key amino acid residues essential for binding with DNA, which further revealed the molecular mechanism of the interaction between Ms6564 and its target DNA. This would help us to further understanding of the function of Ms6564.