Simultaneous binding of two DNA duplexes to the NtrC-enhancer complex studied by two-color fluorescence cross-correlation spectroscopy

The transcription activator protein NtrC (nitrogen regulatory protein C, also termed NRI) 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 DNA sequence, interaction of this complex with promoter-bound RNAP.sigma(54) via DNA looping, and hydrolysis of ATP. Here it is demonstrated by two-color fluorescence cross-correlation spectroscopy measurements of 6-carboxyfluorescein and 6-carboxy-X-rhodamine-labeled DNA oligonucleotide duplexes that the NtrC-P complex can bind two DIVA duplexes simultaneously. This suggests a model for the conformation of the looped intermediate that is formed between NtrC-P and RNAP.sigma(54) at the glnAp2 promoter during the activation process.