A central step in promoter activation by RNA polymerase (RNAP) is the localized separation of the DNA strands to form the transcription bubble. We have used potassium permanganate footprinting to monitor DNA strand-separation by the Bacillus subtilis sigmaD RNAP at the strong promoter, Phag, directing transcription of flagellin. The susceptibility of individual thymine bases to permanganate oxidation is influenced by temperature, Mg2+, nucleotides, and the RNAP delta subunit. In the absence of delta, sigmaD RNAP establishes a partially opened complex even at 0 degrees C with permanganate reactivity localized between -11 and -4 (RP(-4)). The region of strand separation expands to near -1 at 20 degrees C (RP(-1)) and to +3 at 40 degrees C (RP(+3)). The delta subunit inhibits the downstream propagation of the transcription bubble and thereby increases the concentration of early intermediates in the melting pathway. Indeed, E delta sigmaD forms a distinct nucleated complex (RPn) at 0 degrees C with a structural distortion localized to an AT base step within the -10 element. We propose a model for promoter melting in which strand separation nucleates within the conserved -10 consensus and subsequently propagates downstream. Mg2+ and nucleoside triphosphates (NTPs) favor the downstream propagation of the transcription bubble and strongly stimulate the RP(-1) to RP(+3) conversion. The NTP effects are apparently mediated by binding of substrate to the initiating NTP site: purines are more effective than pyrimidines and GMP alone can greatly increase the level of DNA-melting. The binding of substrates, but not Mg2+ alone, can effectively overcome the anti-melting effect of delta.