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Mutational analysis of the highly conserved C‐terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators
Author(s) -
Manzanera Maximino,
Marqués Silvia,
Ramos Juan L
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01749-x
Subject(s) - conserved sequence , helix turn helix , biology , genetics , dna , mutant , transcription (linguistics) , repressor , transcription factor , peptide sequence , gene , linguistics , philosophy
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.