THE RNA-POLYMERASE ALPHA-SUBUNIT CARBOXYL-TERMINAL DOMAIN IS REQUIREDFOR BOTH BASAL AND ACTIVATED TRANSCRIPTION FROM THE ALKA PROMOTER

Expression of the Escherichia coli adaptive response genes (ada, aidB, and alkA) is regulated by the transcriptional activator, Ada, However , the interactions of RNA polymerase and Ada with these promoters diff er. In this report we characterize the interactions of Ada, methylated Ada ((me)Ada), and RNA polymerase at the alkA promoter and contrast t hese interactions with those characterized previously for the ada and aidB promoters, At the alkA promoter, we do not detect the RNA polymer ase alpha subunit-mediated binary complex detected at the uda and aidB promoters, In the presence of either of these two activators, RNA pol ymerase protects the alkA core promoter, including the elements at -35 and -10, and is more efficient in transcription initiation in vitro, RNA polymerase holoenzyme containing the alpha subunit mutation R265A is severely impaired in Ada-independent basal alkA transcription, show s no activation by Ada or (me)Ada, and fails to bind the alkA promoter in vitro. Binding of the purified wild type alpha subunit to alkA was not detected, but a complex of promoter DNA, Ada or (me)Ada, and alph a was observed in gel shift assays, These observations suggest that bo th forms of Ada protein activate alkA transcription by enhancing RNA p olymerase holoenzyme and alpha subunit binding.