DNA-MELTING AT THE BACILLUS-SUBTILIS FLAGELLIN PROMOTER NUCLEATES NEAR -10 AND EXPANDS UNIDIRECTIONALLY

A central step in promoter activation by RNA polymerase (RNAP) is the localized separation of the DNA strands to form the transcription bubb le. We have used potassium permanganate footprinting to monitor DNA st rand-separation by the Bacillus subtilis sigma(D) RNAP at the strong p romoter, P-hag directing transcription of flagellin. The susceptibilit y of individual thymine bases to permanganate oxidation is influenced by temperature, Mg2+, nucleotides, and the RNAP delta subunit. Ln the absence of delta, sigma(D) RNAP establishes a partially opened complex even at 0 degrees C with permanganate reactivity localized between -1 1 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 bubb le and thereby increases the concentration of early intermediates in t he melting pathway. Indeed, E delta sigma(D) forms a distinct nucleate d complex (RP(n)) at 0 degrees C with a structural distortion localize d to an AT base step within the -10 element. We propose a model for pr omoter melting in which strand separation nucleates within the conserv ed -10 consensus and subsequently propagates downstream. Mg2+ and nucl eoside triphosphates (NTPs) favor the downstream propagation of the tr anscription bubble and strongly stimulate the RP(-1) to RP(+3) convers ion. The NTP effects are apparently mediated by binding of substrate t o the initiating NTP site: purines are more effective than pyrimidines and GMP alone can greatly increase the level of DNA-melting. The bind ing of substrates, but not Mg2+ alone, can effectively overcome the an ti-melting effect of delta. (C) 1997 Academic Press Limited.