Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon

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Abstract
In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR–DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR–DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues.