Simultaneous Binding of Two DNA Duplexes to the NtrC−Enhancer Complex Studied by Two-Color Fluorescence Cross-Correlation Spectroscopy

• Published in: Biochemistry (Easton) 2000-03, Vol.39 (9), p.2131-2139
• Main Author: Rippe, Karsten
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Base Sequence; Diffusion; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Enhancer Elements, Genetic; enhancers; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli Proteins; Models, Chemical; Models, Molecular; Molecular Sequence Data; NtrC protein; Nucleic Acid Heteroduplexes - chemistry; Nucleic Acid Heteroduplexes - metabolism; Phosphorylation; PII Nitrogen Regulatory Proteins; Protein Binding; Reproducibility of Results; s@@u54@ factor; Spectrometry, Fluorescence - methods; Trans-Activators; Transcription Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9922190

The transcription activator protein NtrC (nitrogen regulatory protein C, also termed NRI) can catalyze the transition of Escherichia coli RNA polymerase complexed with the σ54 factor (RNAP·σ54) from the closed complex (RNAP·σ54 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·σ54 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·σ54 at the glnAp2 promoter during the activation process.