Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators

Abstract

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so-called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C-terminal end. This conserved region is believed to contain seven α-helices, including two helix-turn-helix (HTH) DNA binding motifs (α 2-T-α 3 and α 5-Tα- 6), connected by a linker α-helix (α 4), and two flanking α-helices (α 1 and α 7). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to ′ lacZ. The analysis revealed that mutations in the α 5-helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the α 6-helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly-290 in the turn between the two helices, Pro-309 located downstream of α 6, and Leu-313, in the small last helix α 7, that seems to play an important role in the activation of RNA-polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.