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 (NR(I)) on its binding properties. Both phosphorylated and unphosphorylated NR(I) bind linearly to a single binding site but cooperatively to two adjacent binding sites. Cooperative binding of NR(I) is severely affected by phosphorylation: half-maximal binding of NR(I)-phosphate is at 20-fold lower concentrations than that of unphosphorylated NR(I). This is more due to a huge increase in the cooperativity constant-which is the strength of interaction between two NR(I) 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 NR(I) is not enough for efficient activation and that activation only occurs at a higher NR(I) concentration. We propose an activation mechanism for NR(I) in which the phosphorylation of NR(I) induces a conformational change in the N-terminal domains of the NR(I)-phosphate dimers, which now interact strongly with each other, leading to a tetramerization of NR(I) upon binding to two adjacent binding sites. We propose that not the phosphorylation of NR(I) itself but rather the tetramerization of NR(I)-phosphate on DNA binding induces the conformational change of the central domain to the active conformation.