ACTIVATION OF THE TRPBA PROMOTER OF PSEUDOMONAS-AERUGINOSA BY TRPI PROTEIN INVITRO

We have developed an in vitro transcription system in which purified TrpI protein and indoleglycerol phosphate (InGP) activate transcription initiation at the trpBA promoter (trpP(B)) and repress initiation at the trpI promoter (trpP(I)) of Pseudomonas aeruginosa. The phenotypes resulting from mutations in the -10 region of both promoters indicate that the -10 region consensus sequence in P. aeruginosa is probably the same as that in Escherichia coli. Furthermore, in the absence of TrpI and InGP, the activities of the two promoters are inversely correlated: down mutations in trpP(I) lead to increased activity of trpP(B), and up mutations in trpP(B) cause a decrease in trpP(I) activity. These results are a consequence of the fact that the two promoters overlap, so that RNA polymerase cannot form open complexes with both promoters simultaneously. Thus, in theory, by preventing RNA polymerase from binding at trpP(I), TrpI protein could indirectly activate trpP(B). However, oligonucleotide-induced mutations that completely inactivate trpP(I) do not relieve the requirement for TrpI and InGP to activate trpP(B). Therefore, activation of trpP(B) is mediated by a direct effect of TrpI on transcription initiation at trpP(B). In addition, the oligonucleotide-induced mutations in trpP(I) alter site II, the weaker of two TrpI binding sites identified in DNase I and hydroxyl radical footprinting studies (M. Chang and I. P. Crawford, Nucleic Acids Res. 18:979-988, 1990). Since these mutations prevent full activation of trpP(B), we conclude that specific base pairs in site II are required for activation.