In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell. Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face. E sigma 54 protects two major grooves of DNA approximately 60 base pairs downstream at the glnAp2 promoter and primarily on the opposite helix face. Experiments using potassium permanganate to probe open complex formation in vivo demonstrate that NRI is absolutely required for E sigma 54 to open the promoter DNA. Together, the dimethyl sulfate and permanganate studies verify [Reitzer, L. J., Bueno, R., Cheng, W. D., Abrams, S. A., Rothstein, D. M., Hunt, T. P., Tyler, B. & Magasanik, B. (1987) J. Bacteriol. 169, 4279-4284] that E sigma 54 occupies the glnAp2 promoter in a closed complex in vivo even in the presence of excess nitrogen and the absence of NRI. Furthermore, the slow step in transcriptional activation is shown to be an NRI-dependent conformational change in the downstream promoter DNA, which results in DNA melting. These observations place interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.