ACTIVATION OF THE CATBCA PROMOTER - PROBING THE INTERACTION OF CATR AND RNA-POLYMERASE THROUGH IN-VITRO TRANSCRIPTION

The soil bacterium Pseudomonas putida is capable of degrading many aro matic compounds, including benzoate, through catechol as an intermedia te. The catabolism of catechol is mediated by the catBCA operon, whose induction requires the pathway intermediate cis,cis-muconate as an in ducer and the regulatory protein, CatR. CatR also regulates the plasmi d-borne pheBA operon of P. putida PaW85, which is involved in phenol c atabolism. We have used an in vitro transcription system to study the roles of CatR cis,cis-muconate, Escherichia call RNA polymerase, and p romoter sequences in expression of the cat and phe operons. The assay confirmed the requirement of both CatR and cis,cis-muconate for transc ript formation. We also examined the in vitro transcription of three s ite-directed mutants of the catBCA promoter; the results obtained comp ared favorably with previous in vivo data. The requirement of the alph a subunit of RNA polymerase for expression of the catBCA and the pheBA transcripts was also examined. The C-terminal region of the alpha sub unit of RNA polymerase has been implicated in direct protein-protein c ontact with transcriptional regulatory proteins and/or direct contact with the DNA. We show that the carboxyl terminus of the alpha subunit is required for the expression of the catBCA and the pheBA operons bec ause RNA polymerases with truncated alpha subunits were deficient in a ctivation. Further experiments demonstrated the arginine at position 2 65 and the asparagine at position 268 of the alpha subunit as possible amino acids involved in activation. On the basis of these and previou s results, we propose a model to explain the interaction of the differ ent regulatory components leading to CatR-dependent activation of the catBCA operon.