Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators.
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 alpha-helices, including two helix-turn-helix (HTH) DNA binding motifs (alpha(2)-T-alpha(3) and alpha(5)-Talpha-(6)), connected by a linker alpha-helix (alpha(4)), and two flanking alpha-helices (alpha(1) and alpha(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 alpha(5)-helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the alpha(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 alpha(6), and Leu-313, in the small last helix alpha(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.
Manzanera
M
M
CSIC, Estación Experimental del Zaidín, Departamento de Bioquímica y Biología Molecular y Celular de Plantas, Apdo. 419, 18080, Granada, Spain.
Marqués
S
S
Ramos
J L
JL
D000595:Amino Acid Sequence; D051777:AraC Transcription Factor; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D016958:Pseudomonas putida / Q000235:genetics*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics*; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C080002:MarA protein, E coli; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C078720:XylS protein, Pseudomonas putida