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

The transcription activator protein NtrC (nitrogen regulatory protein C) ca n catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma 54 factor (RNAP . sigma(54)) from the closed complex (RNAP . sig ma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of a n octameric NtrC-P complex at the enhancer sequence, interaction of this co mplex with promoter-bound RNAP sigma 54 via DNA looping, and hydrolysis of Am. We have used this system to study the influence of the DNA conformation on the transcription activation rate in single-round transcription experim ents with superhelical plasmids as well as linearized templates. Most of th e templates had an intrinsically curved DNA sequence between the enhancer a nd the promoter and differed with respect to the location of the curvature and the distance between the two DNA sites. The following results were obta ined: (i) a ten- to 60-fold higher activation rate was observed with the su perhelical templates as compared to the linearized conformation; (ii) the p resence of an intrinsically curved DNA sequence increased the activation ra te 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 vari ed 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 super helical templates studied. The results were compared with theoretical calcu lations 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 comple x formation was developed. (C) 2000 Academic Press.