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.

Schulz A ，Langowski J ，Rippe K 《JOURNAL OF MOLECULAR BIOLOGY》

Association states of the transcription activator protein NtrC from E. coli determined by analytical ultracentrifugation.

The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of E. coli RNA polymerase complexed with the sigma54 factor (RNAP.sigma54) from the closed complex (RNAP.sigma54 bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC, assembly of a multimeric NtrC complex at the enhancer DNA sequence, interaction of this complex with promoter bound RNAP. sigma54 via DNA looping, and hydrolysis of ATP. We have used analytical ultracentrifugation to study the different NtrC association states and to derive hydrodynamic models for the conformation of the various NtrC species. The following results were obtained. (i) The unphosphorylated wild-type protein formed a dimer with a measured molecular weight of 102(+/-3) kDa, which compares to a calculated molecular weight of 54 kDa for a monomer (concentration range studied 2 to 8 microM NtrC monomer). (ii) In the unphosphorylated state one NtrC dimer was bound to one binding site as determined with DNA oligonucleotide duplexes containing one or two binding sites (concentration range studied 50 to 1000 nM NtrC dimer). (iii) The data obtained at protein concentrations that were below the concentration of binding sites indicate that binding to the DNA duplex with two binding sites occurred with essentially no cooperativity. The experiments were conducted in the absence of ATP. (iv) The phosphorylated protein formed a specific complex at the DNA duplex with the enhancer sequence (two NtrC binding sites) that consisted of four dimers (concentration range studied 100 to 1000 nM NtrC dimer). (v) The formation of this octameric complex was highly cooperative, and the data suggest that two DNA strands could bind simultaneously to this complex. (vi) From the sedimentation data a model was derived in which the NtrC dimer adopts a V shaped structure with the DNA binding domains being located at the bottom and the two receiver domains at the top of the V. In this conformation higher order NtrC complexes can be stabilized by interaction between the phosphorylated receiver domain and the central activation domain of different NtrC dimers.

Rippe K ，Mücke N ，Schulz A 《JOURNAL OF MOLECULAR BIOLOGY》

Beta subunit residues 186-433 and 436-445 are commonly used by Esigma54 and Esigma70 RNA polymerase for open promoter complex formation.

During transcription initiation by DNA-dependent RNA polymerase (RNAP) promoter DNA has to be melted locally to allow the synthesis of RNA transcript. Localized melting of promoter DNA is a target for genetic regulation and is poorly understood at the molecular level. The Escherichia coli RNAP holoenzyme is a six-subunit (alpha(2)betabeta'omegasigma; Esigma) protein complex. The sigma subunit is directly responsible for promoter recognition and contributes to localized DNA melting. Mutations in the beta subunit have profound effects on promoter melting by Esigma70. The sigma54 subunit is a representative of an unrelated class of the sigma subunits. Here, we determined whether mutations in the beta subunit that affect late stages of promoter complex formation by Esigma70 also influence promoter complex formation by the enhancer-dependent Esigma54. Analyses of in vitro defects in promoter complex formation and transcription initiation exhibited by mutant Esigma54 suggest that during promoter complex formation by Esigma54 and Esigma70 a common set of beta subunit sequences is used. Late stages of promoter complex formation and localized melting of promoter DNA by Esigma70 and Esigma54 thus proceed through a common pathway.

Wigneshweraraj SR ，Nechaev S ，Severinov K ，Buck M ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Isomerization of a binary sigma-promoter DNA complex by transcription activators.

Cannon WV ，Gallegos MT ，Buck M 《-》

Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting.

Young BA ，Gruber TM ，Gross CA 《-》