DNASE-I FOOTPRINTING, DNA BENDING AND IN-VITRO TRANSCRIPTION ANALYSESOF CLCR AND CATR INTERACTIONS WITH THE CLCABD PROMOTER - EVIDENCE OF A CONSERVED TRANSCRIPTIONAL ACTIVATION MECHANISM

In Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolize d to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous tr anscriptional activators CatR and ClcR. Previous studies have demonstr ated that in addition to sequence similarities, CatR and ClcR share fu nctional similarities which allow catR to complement clcR. In this stu dy, we demonstrate that CatR activates the clcABD promoter in vitro wi thout inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA-bending analyses demonstrate that CatR bi nds to and bends the clcABD promoter to the same angle as does ClcR pl us its inducer, 2-chloromuconate. This implies that CatR binds to the de promoter in its active conformation. Transcription of the clcABD pr omoter by the a-subunit truncation mutant (alpha-235) of RNA polymeras e was sharply reduced, indicating that the alpha-subunit C-terminal do main is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.