Several activators of sigma 70 holoenzyme whose binding sites lie upstream of the -35 region of promoters require the C-terminal region of the alpha subunit of RNA polymerase to activate transcription. (These are among class I activators, which require the C-terminal region of the alpha subunit for transcription activation.) Because transcription by sigma 54 holoenzyme universally depends upon activators whose binding sites lie well upstream (or downstream) of promoters, we determined whether the C-terminal region of the alpha subunit was also required for transcription from the sigma 54-dependent promoter for the glnA operon. Nitrogen regulatory protein C-dependent activation from the glnA promoter remained good when RNA polymerases containing C-terminal truncations of the alpha subunit were employed. This was also the case for nitrogen fixation protein A-dependent activation if a nitrogen fixation protein A-binding site was appropriately placed upstream of the glnA promoter. These results lead to the working hypothesis (as yet untested) that activators of sigma 54 holoenzyme, which appear to make direct physical contact with the polymerase to catalyze a change in its conformation, activate the sigma 54 holoenzyme by contacting the sigma subunit rather than the alpha subunit of the core enzyme.