The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase alpha subunit C-terminal domain. Using preparations of E. coli RNA polymerase holoenzyme, specifically labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase alpha subunit in transcriptionally competent complexes at a class II FNR-dependent promoter. We conclude that one alpha subunit C-terminal domain binds immediately upstream of FNR, and that its position and orientation is the same as at similar promoters dependent on CRP, another E. coli transcription activator that is related to FNR. In complementary experiments, we show that the second alpha subunit C-terminal domain of RNA polymerase can be repositioned by upstream-bound CRP, but not by upstream-bound FNR.