Recognition of binding sites I and II by the TrpI activator protein of Pseudomonas aeruginosa: efficient binding to both sites requires InGP even when site II is replaced by site I

TrpI protein, the activator of transcription of the trpBA operon of three s pecies of fluorescent Pseudomonads, bends the DNA when it forms either of t wo well-characterized complexes with the trpBA regulatory region. In comple x 1, TrpI is bound only to its strong binding site (site I), whereas in com plex 2, which is required for activation of the trpBA promoter, TrpI is bou nd both to site I and to the weaker site II. Indoleglycerol phosphate (InGP ) strongly stimulates formation of complex 2 and is required for activation . The present study focuses on the binding of TrpI to DNA containing a dupl ication of site I and the effect of the duplication on TrpI-induced DNA ben ding. We find that even on DNA containing a tandem (direct or inverted) dup lication of site I, the formation of DNA-TrpI complexes with both sites occ upied is strongly stimulated by InGP. Thus, even when TrpI binding to two a djacent sites needs not be cooperative, InGP significantly promotes the for mation of complex 2. Gel binding data indicate that InGP can have several e ffects: (1) TrpI molecules bound to either of two adjacent strong binding s ites appear to interfere with binding to the other site; InGP relieves this apparent interference. (2) InGP increases the intrinsic affinity of TrpI f or sites I and II and/or enhances cooperative TrpI binding to adjacent DNA sites. Furthermore, a third molecule of TrpI can form a footprint adjacent to the duplication on DNA containing a direct (but not inverted) repeat of site I, indicating that TrpI bound to site I is oriented asymmetrically in spite of the quasi-symmetry of the binding site. The calculated bending ang le for DNA in complex 2 is increased by approximately 20 degrees when site I is substituted in either orientation for site II; thus, on DNA containing a site I duplication, the bending angle of complex 2 is nearly twice that of complex 1. (C) 1998 Elsevier Science B.V. All rights reserved.