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 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.
Rippe
K
K
Deutsches Krebsforschungszentrum, Abteilung Biophysik der Makromoleküle, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Karsten.Rippe@dkfz-heidelberg.de
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins; D010766:Phosphorylation; D011485:Protein Binding; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D015534:Trans-Activators; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli