XylS activator and RNA polymerase binding sites at the Pm promoter overlap

Abstract

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 6GGNTA-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°, 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.