A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation.
A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation.
复制标题
OmpR C 末端的单个氨基酸取代会改变 DNA 识别和磷酸化。
DOI：
10.1006/jmbi.2000.3809
复制
发表时间：
2000
影响因子：
5.6
通讯作者：
Kenney,LJ
中科院分区：
文献类型：
--
作者：
Tran,VK;Oropeza,R;Kenney,LJ
在细菌和低等真核生物中，对环境变化的适应通常是由双组分调控系统介导的。例如，这些系统为趋化性、氮和磷的调节以及适应渗透胁迫提供了基础。在大肠杆菌中，传感器激酶EnvZ检测渗透环境的变化，并磷酸化反应调节因子OmpR。磷酸化的OmpR结合到孔蛋白基因OmpF和OMPC的调节区，并改变它们的表达。最近的证据表明，OmpR发挥着全球调节作用，除了调节孔蛋白基因外，还调节其他基因。在这项研究中，我们鉴定了一个先前分离的OmpR2突变体(V203M)，它结构性地激活了OmpF，但无法表达OMPC。由于该替换位于DNA结合结构域的C末端，因此人们认为该替换不会影响OmpR的N末端结构域的磷酸化。我们的结果表明，这种取代完全消除了一个小的磷酸供体--乙酰磷酸的磷酸化，但不能消除激酶EnvZ的磷酸化。与野生型相比，突变的OmpR改变了去磷酸化动力学，改变了与OmpF和OMPC位点的结合亲和力。因此，C端DNA结合域中的单个氨基酸替换对N端磷酸化结构域有显著影响。最引人注目的是，我们已经确定OMPC的OmpR结合位点发生了单一碱基变化，从而恢复了突变体的高亲和力结合活性。我们在一个孔蛋白基因表达模型的背景下解释了我们的结果。
In bacteria and lower eukaryotes, adaptation to changes in the environment is often mediated by two-component regulatory systems. Such systems provide the basis for chemotaxis, nitrogen and phosphate regulation and adaptation to osmotic stress, for example. In Escherichia coli, the sensor kinase EnvZ detects a change in the osmotic environment and phosphorylates the response regulator OmpR. Phospho-OmpR binds to the regulatory regions of the porin genes ompF and ompC, and alters their expression. Recent evidence suggests that OmpR functions as a global regulator, regulating additional genes besides the porin genes. In this study, we have characterized a previously isolated OmpR2 mutant (V203M) that constitutively activates ompF and fails to express ompC. Because the substitution was located in the C-terminal DNA-binding domain, it had been assumed that the substitution would not affect phosphorylation of the N-terminal domain of OmpR. Our results indicate that this substitution completely eliminates phosphorylation by a small phosphate donor, acetyl phosphate, but not phosphorylation by the kinase EnvZ. The mutant OmpR has altered dephosphorylation kinetics and altered binding affinities to both ompF and ompC sites compared to the wild-type. Thus, a single amino acid substitution in the C-terminal DNA-binding domain has dramatic effects on the N-terminal phosphorylation domain. Most strikingly, we have identified a single base change in the OmpR binding site of ompC that restores high-affinity binding activity by the mutant. We interpret our results in the context of a model for porin gene expression.