The effect of the DNA conformation on the rate of NtrC activated transcription of Escherichia coli RNA polymerase.sigma(54) holoenzyme.

The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma 54 factor (RNAP.sigma(54)) from the closed complex (RNAP.sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer sequence, interaction of this complex with promoter-bound RNAP.sigma(54) via DNA looping, and hydrolysis of ATP. We have used this system to study the influence of the DNA conformation on the transcription activation rate in single-round transcription experiments with superhelical plasmids as well as linearized templates. Most of the templates had an intrinsically curved DNA sequence between the enhancer and the promoter and differed with respect to the location of the curvature and the distance between the two DNA sites. The following results were obtained: (i) a ten- to 60-fold higher activation rate was observed with the superhelical templates as compared to the linearized conformation; (ii) the presence of an intrinsically curved DNA sequence increased the activation rate of linear templates about five times; (iii) no systematic effect for the presence and/or location of the inserted curved sequence was observed for the superhelical templates. However, the transcription activation rate varied up to a factor of 10 between some of the constructs. (iv) Differences in the distance between enhancer and promoter had little effect for the superhelical templates studied. The results were compared with theoretical calculations for the dependence of the contact probability between enhancer and promoter expressed as the molar local concentration j(M). A correlation of j(M) with the transcription activation rate was observed for values of 10(-8) M<j(M)<10(-6) M and a kinetic model for NtrC-P-catalyzed open complex formation was developed. Copyright 2000 Academic Press.