Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis.
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 μm) 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.
Council of Scientific and Industrial Research, India, Institute of Microbial Technology, Chandigarh, India.
Sushma
S
Anand
Vijay
V
Singh
Appu Kumar
AK
Singh
Monica
M
Mittal
Manish
M
Datt
Bala
B
Subramani
Sangaralingam
S
Kumaran
D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D020349:Surface Plasmon Resonance
D004269:DNA, Bacterial; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein