Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription.
We studied the effect of phosphorylation of nitrogen regulator I (NRI) on its binding properties. Both phosphorylated and unphosphorylated NRI bind linearly to a single binding site but cooperatively to two adjacent binding sites. Cooperative binding of NRI is 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 NRI dimers--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 NRI is not enough for efficient activation and that activation only occurs at a higher NRI concentration. We propose an activation mechanism for NRI in which the phosphorylation of NRI induces 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 NRI upon binding to two adjacent binding sites. We propose that not the phosphorylation of NRI itself but rather the tetramerization of NRI-phosphate on DNA binding induces the conformational change of the central domain to the active conformation.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
V
V
Weiss
F
F
Claverie-Martin
B
B
Magasanik
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D051922:PII Nitrogen Regulatory Proteins; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D051922:PII Nitrogen Regulatory Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; D005974:Glutamate-Ammonia Ligase