Phosphorylation of Nitrogen Regulator I of Escherichia coli Induces Strong Cooperative Binding to DNA Essential for Activation of Transcription

• Published in: Proceedings of the National Academy of Sciences - PNAS 1992-06, Vol.89 (11), p.5088-5092
• Main Authors: Weiss, Verena, Claverie-Martin, Felix, Magasanik, Boris
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Biochemistry; Biological and medical sciences; Cooperation; Dimers; DNA; DNA-Binding Proteins - physiology; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Glutamate-Ammonia Ligase - genetics; Ligands; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Molecules; Nitrogen; Oligodeoxyribonucleotides - chemistry; Phosphates; Phosphorylation; PII Nitrogen Regulatory Proteins; Plasmids; Protein Binding; Regulator genes; Regulatory Sequences, Nucleic Acid; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.11.5088

We studied the effect of phosphorylation of nitrogen regulator I (NRI) on its binding properties. Both phosphorylated and unphosphorylated NRIbind linearly to a single binding site but cooperatively to two adjacent binding sites. Cooperative binding of NRIis severely affected by phosphorylation: half-maximal binding of NRI-phosphate is at 20-fold lower concentrations than that of unphosphorylated NRI. This is more due to a huge increase in the cooperativity constant-which is the strength of interaction between two NRIdimers-than to an increase in the microscopic binding constant which is the binding affinity to a single binding site. In vitro transcription and DNA footprinting experiments showed that occupation of a single binding site by NRIis not enough for efficient activation and that activation only occurs at a higher NRIconcentration. We propose an activation mechanism for NRIin which the phosphorylation of NRIinduces a conformational change in the N-terminal domains of the NRI-phosphate dimers, which now interact strongly with each other, leading to a tetramerization of NRIupon binding to two adjacent binding sites. We propose that not the phosphorylation of NRIitself but rather the tetramerization of NRI-phosphate on DNA binding induces the conformational change of the central domain to the active conformation.