Leucines 193 and 194 at the N-terminal domain of the XylS protein, the positive transcriptional regulator of the TOL meta-cleavage pathway, are involved in dimerization.
Members of the AraC/XylS family of transcriptional regulators are usually organized in two domains: a conserved domain made up of 100 amino acids and frequently located at the C-terminal end, involved in DNA binding; and an N-terminal nonconserved domain involved in signal recognition, as is the case for regulators involved in the control of carbon metabolism (R. Tobes and J. L. Ramos, Nucleic Acids Res. 30:318-321, 2002). The XylS protein, which is extremely insoluble, controls expression of the meta-cleavage pathway for alkylbenzoate metabolism. We fused the N-terminal end of XylS to the maltose-binding protein (MBP) in vitro and found in glutaraldehyde cross-linking assays that the protein dimerized. Experiments with a chimeric N-terminal XylS linked to a 'LexA protein showed that the dimer was stabilized in the presence of alkylbenzoates. Sequence alignments with AraC and UreR allowed us to identify three residues, Leu193, Leu194, and Ile205, as potentially being involved in dimerization. Site-directed mutagenesis of XylS in which each of the above residues was replaced with Ala revealed that Leu193 and Leu194 were critical for activity and that a chimera in which LexA was linked to the N terminus of XylSLeu193Ala or XylSLeu194Ala was not functional. Dimerization of the chimeras MBP-N-XylSLeu193Ala and MBP-N-XylSLeu194Ala was not observed in cross-linking assays with glutaraldehyde.
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008 Granada, Spain.
Raquel
R
Ruíz
Silvia
S
Marqués
Juan L
JL
Ramos
D000595:Amino Acid Sequence; D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D004268:DNA-Binding Proteins; D019281:Dimerization; D005976:Glutaral / Q000737:chemistry; D007930:Leucine; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques
D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C119767:UreR protein, bacteria; C078720:XylS protein, Pseudomonas putida; D007930:Leucine; D005976:Glutaral