Chen P, Reitzer L J
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson 75083-0688, USA.
J Bacteriol. 1995 May;177(9):2490-6. doi: 10.1128/jb.177.9.2490-2496.1995.
Activation by the prokaryotic activator nitrogen regulator I (NRI, or NtrC) of Escherichia coli requires an interaction between two NRI dimers. ATP-dependent phosphorylation stimulates this tetramerization, which can be detected as cooperative binding to DNA. A polypeptide containing only the DNA-binding carboxyl-terminal domain has been previously shown to bind noncooperatively to DNA. Our primary purpose was to determine whether the highly conserved N-terminal domain or the ATP-binding central domain is required for cooperative DNA binding. Because ATP was present in the experiments that showed that phosphorylation enhances cooperative bindings, it is possible that ATP and not phosphorylation stimulated cooperative binding. Our secondary purpose was to separately assess the effects of ATP and phosphorylation on cooperative binding. We showed that a variant with a deletion of the central domain, NRI-(delta 143-398), binds cooperatively as well as unphosphorylated wild-type NRI, implying that the N-terminal domain mediates phosphorylation-independent cooperative binding. Phosphorylation of NRI-(delta 143-398) did not further stimulate this binding, suggesting that the ATP-binding central domain may be required for the phosphorylation-dependent enhancement. Cooperative binding was enhanced by either acetyl-phosphate-dependent (i.e., ATP-independent) phosphorylation of NRI or the specific binding of ATP to the central domain. Their effects were not additive, a finding which is consistent with the interpretation that each promotes a similar dimer-dimer interaction. We discuss these results within the context of the hypothesis that the highly conserved N-terminal domain mediates phosphorylation-independent cooperativity and the central domain is required for cooperativity stimulated by ATP binding or phosphorylation.
大肠杆菌的原核激活因子氮调节蛋白I（NRI，即NtrC）的激活需要两个NRI二聚体之间相互作用。ATP依赖性磷酸化刺激这种四聚化，可检测为与DNA的协同结合。先前已表明，仅包含DNA结合羧基末端结构域的多肽与DNA的结合是非协同性的。我们的主要目的是确定协同DNA结合是否需要高度保守的N末端结构域或ATP结合中央结构域。因为在表明磷酸化增强协同结合的实验中存在ATP，所以有可能是ATP而非磷酸化刺激了协同结合。我们的次要目的是分别评估ATP和磷酸化对协同结合的影响。我们发现，缺失中央结构域的变体NRI-（δ143-398）与未磷酸化的野生型NRI一样具有协同结合能力，这意味着N末端结构域介导了不依赖磷酸化的协同结合。NRI-（δ143-398）的磷酸化并未进一步刺激这种结合，这表明ATP结合中央结构域可能是磷酸化依赖性增强所必需的。NRI的乙酰磷酸依赖性（即不依赖ATP）磷酸化或ATP与中央结构域的特异性结合均可增强协同结合。它们的作用并非相加性的，这一发现与以下解释一致：每种作用都促进了类似的二聚体-二聚体相互作用。我们在以下假设的背景下讨论这些结果：高度保守的N末端结构域介导不依赖磷酸化的协同性，而中央结构域是ATP结合或磷酸化刺激的协同性所必需的。
