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Updated: Aug 10, 2026
High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Published on: February 7, 2019
Simultaneous binding of two DNA duplexes to the NtrC-enhancer complex studied by two-color fluorescence
1Deutsches Krebsforschungszentrum, Abteilung Biophysik der Makromoleküle, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Karsten.Rippe@dkfz-heidelberg.de
Abstract:
The transcription activator protein NtrC (nitrogen regulatory protein C, also termed NR(I)) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma(54) factor (RNAP x sigma(54)) from the closed complex (RNAP x 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 x 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 DNA 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.
