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 s54 factor (RNAP s54) from the closed complex (RNAP s54 bound at the promoter) to the open complex (melting of the promoter DNA). This pro-cess involves phosphorylation of NtrC, assembly of a multimeric NtrC complex at the enhancer DNA sequence, interaction of this complex with promoter bound RNAP s54 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 confor-mation 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 calcu-lated molecular weight of 54 kDa for a monomer (concentration range studied 2 to 8 mM NtrC monomer). (ii) In the unphosphorylated state one NtrC dimer was bound to one binding site as determined with DNA oli