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 XyIS 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 [Gonzalez-Perez et al. (1999) J. Biol. Chem. 274, 2286-2290]. An intrinsic bending of 40degrees, which is not essential for transcription, is centered at position -43. We have determined the potential overlap between the XyIS and RNA polymerase binding sites. The insertion of 2 or more lip between C and T at positions -37 and -36 abolished transcription activation by the wild-type XyIS and by XyISS2291, a mutant with increased affinity for the XyIS binding sites. In contrast, a 1-bp insertion at -37 was permissible, although when in addition to the I-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 XyISS2291. 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. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.