Functional definition of the two effector binding sites, the oligomerization and DNA binding domains of the Bacillus subtilis LysR-type transcriptional regulator AlsR.

Abstract

The LysR-type transcriptional regulator (LTTR) AlsR from Bacillus subtilis activates the transcription of the alsSD operon encoding enzymes for acetoin formation in response to the presence of acetate. The structural basis for effector binding, oligomerization, DNA binding, higher ordered complex formation, DNA bending and transcriptional control by B. subtilis AlsR was functionally characterized. The binding of two molecules of acetate per molecule AlsR was determined. Acetate-dependent transcription complex formation was observed. A structural model of AlsR was used to identify the amino acid residues V98, S100, H147 of the binding site 1, which were experimentally verified. The second binding site formed by T193, V194, A196, T201 and L202 mediated high acetate responsive induction. Residues L124, E225 Q74, I79 and R111 contributed to dimerization of AlsR. A22, Q29, P30, S33, K37, L39, E46, R50 and R53 of the winged helix-turn-helix motif were important for promoter recognition. The DNA binding domain alone dimerized and effectively bound the promoter. The LTTR promoter elements RBS and ABS had to be localized on the same site of the DNA. Higher ordered complex formation resulted in bending of promoter DNA and transcriptional activation.