%A Sushma Anand
%A Vijay Singh
%A Appu Kumar Singh
%A Monica Mittal
%A Manish Datt
%A Bala Subramani
%A Sangaralingam Kumaran
%O Copyright of this article belongs to John Wiley and sons.
%J The FEBS journal
%T Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis.
%X Fatty acids play critical role in the survival and virulence of Mycobacterium tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia coli, which has single enzyme. However, the mechanisms by which the expression of these multiple Fad genes is regulated remain uncharacterized. We characterized the DNA- and ligand-binding properties of a putative tetracycline repressor family regulator, named Fad35R, located upstream of the Fad35 gene and ScoA-citE operon. We identified a palindromic regulatory motif upstream of Fad35 and characterized the binding of Fad35R to this motif. Equilibrium binding studies show that Fad35R binds to this motif with high affinity (K(d) ? 0.033 ?m) and the specificity of binding was confirmed by an electromobility gel shift assay. Kinetic studies indicate that faster association (k(a,avg) ? 5.4 ? 10(4) m(-1) ? s(-1)) and slower dissociation rates (k(d,avg) ? 5.84 ? 10(-4) s(-1)) confer higher affinity. The affinity for the promoter is maximum at 300 mm NaCl but decreases rapidly beyond this range. Ligand-binding studies indicate that Fad35R binds specifically to tetracycline and also binds to fatty acid derivatives. The promoter-binding affinity is decreased significantly in the presence of palmityl-CoA, suggesting that Fad35R can sense the levels of activated fatty acids and alter its DNA-binding activity. Our results suggest that Fad35R may be the functional homologue of FadR and controls the expression of genes in a metabolite-dependent manner.
%N 17
%K  fatty acid;protein?DNA interactions; surface plasmon resonance; TetR family; transcriptional regulation
%P 3214-28
%V 279
%D 2012
%I John and Wiley
%L open1307