XylS activator and RNA polymerase binding sites at the Pm promoter overlap.
Transcription from the TOL plasmid meta-cleavage pathway operon, Pm, depends on the XylS protein being activated by a benzoate effector. The XylS binding sites are two imperfect 5'-TGCAN(6)GGNTA-3' direct repeats located between positions -70/-56 and -49/-35 [González-Pérez et al. (1999) J. Biol. Chem. 274, 2286-2290]. An intrinsic bending of 40 degrees, which is not essential for transcription, is centered at position -43. We have determined the potential overlap between the XylS and RNA polymerase binding sites. The insertion of 2 or more bp between C and T at positions -37 and -36 abolished transcription activation by the wild-type XylS and by XylSS229I, a mutant with increased affinity for the XylS binding sites. In contrast, a 1-bp insertion at -37 was permissible, although when in addition to the 1-bp insertion at -37 the mutant promoter had a point mutation at the XylS binding site (C-47-->T), transcription was abolished with the wild-type XylS protein, but not with XylSS229I. The overlap between the proximal XylS binding site and the -35 region recognized by RNA polymerase at positions -35 and -36 appears to be critical for transcription.
González-Pérez
M Mar
MM
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, Granada, Spain.
Marqués
Silvia
S
Domínguez-Cuevas
Patricia
P
Ramos
Juan L
JL
D001426:Bacterial Proteins; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D016958:Pseudomonas putida; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida; D012321:DNA-Directed RNA Polymerases