Weiss V, Claverie-Martin F, Magasanik B
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5088-92. 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 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.
我们研究了氮调节因子I（NRI）磷酸化对其结合特性的影响。磷酸化和未磷酸化的NRI均线性结合至单个结合位点，但协同结合至两个相邻的结合位点。NRI的协同结合受到磷酸化的严重影响：磷酸化NRI的半数最大结合浓度比未磷酸化的NRI低20倍。这更多是由于协同常数大幅增加（协同常数是两个NRI二聚体之间的相互作用强度），而非微观结合常数增加（微观结合常数是对单个结合位点的结合亲和力）。体外转录和DNA足迹实验表明，NRI占据单个结合位点不足以实现有效激活，且激活仅在较高的NRI浓度下发生。我们提出了一种NRI的激活机制，其中NRI的磷酸化诱导磷酸化NRI二聚体N端结构域发生构象变化，这些结构域现在彼此强烈相互作用，导致NRI在结合到两个相邻结合位点时发生四聚化。我们提出，不是NRI本身的磷酸化，而是磷酸化NRI在DNA结合时的四聚化诱导中央结构域转变为活性构象。
