title: Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants.
abstract: The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNA-polymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the -40 to -70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position -42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/RNA polymerase interactions. Methylation of T-42 was highest in cells bearing XylS and in the presence of an effector. Gs in the -47 to -61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between -41 and -78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN6GGNCA. These motifs cover the -70 to -56 and the -49 to -35 regions. Single point mutations revealed that nucleotides located at -49 to -46 and at -59, -60, -62, and -70 are the most critical for appropriate XylS-Pm interactions.
authors.affiliation: Department of Biochemistry, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado de Correos 419, E-18008 Granada, Spain.
authors.forename: M M
authors.initials: MM
authors.lastname: González-Pérez
authors.forename: J L
authors.initials: JL
authors.lastname: Ramos
authors.forename: M T
authors.initials: MT
authors.lastname: Gallegos
authors.forename: S
authors.initials: S
authors.lastname: Marqués
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D019175:DNA Methylation; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D020080:Tandem Repeat Sequences; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida; D004247:DNA; D012321:DNA-Directed RNA Polymerases