Department of NMR-based Structural Biology, Max Planck Institute for iophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
J Biol Chem. 2012 Aug 10;287(33):27731-42. doi: 10.1074/jbc.M112.388850. Epub 2012 Jun 21.
The control of several catabolic operons in bacteria by transcription antitermination is mediated by RNA-binding proteins that consist of an RNA-binding domain and two reiterated phosphotransferase system regulation domains (PRDs). The Bacillus subtilis GlcT antitermination protein regulates the expression of the ptsG gene, encoding the glucose-specific enzyme II of the phosphotransferase system. In the absence of glucose, GlcT becomes inactivated by enzyme II-dependent phosphorylation at its PRD1, whereas the phosphotransferase HPr phosphorylates PRD2. However, here we demonstrate by NMR analysis and mass spectrometry that HPr also phosphorylates PRD1 in vitro but with low efficiency. Size exclusion chromatography revealed that non-phosphorylated PRD1 forms dimers that dissociate upon phosphorylation. The effect of HPr on PRD1 was also investigated in vivo. For this purpose, we used GlcT variants with altered domain arrangements or domain deletions. Our results demonstrate that HPr can target PRD1 when this domain is placed at the C terminus of the protein. In agreement with the in vitro data, HPr exerts a negative control on PRD1. This work provides the first insights into how specificity is achieved in a regulator that contains duplicated regulatory domains with distinct dimerization properties that are controlled by phosphorylation by different phosphate donors. Moreover, the results suggest that the domain arrangement of the PRD-containing antitermination proteins is under selective pressure to ensure the proper regulatory output, i.e. transcription antitermination of the target genes specifically in the presence of the corresponding sugar.
细菌中几种分解代谢操纵子的转录终止抑制控制是由 RNA 结合蛋白介导的，这些蛋白由 RNA 结合域和两个重复的磷酸转移酶系统调节域（PRD）组成。枯草芽孢杆菌 GlcT 终止抑制蛋白调节编码磷酸转移酶系统葡萄糖特异性酶 II 的 ptsG 基因的表达。在没有葡萄糖的情况下，GlcT 会被酶 II 依赖性磷酸化在其 PRD1 上失活，而磷酸转移酶 HPr 则磷酸化 PRD2。然而，在这里我们通过 NMR 分析和质谱分析表明，HPr 也可以在体外但效率较低地磷酸化 PRD1。分子筛层析表明，非磷酸化的 PRD1 形成二聚体，在磷酸化后解离。还在体内研究了 HPr 对 PRD1 的影响。为此，我们使用了具有改变的结构域排列或结构域缺失的 GlcT 变体。我们的结果表明，当该结构域位于蛋白质的 C 末端时，HPr 可以靶向 PRD1。与体外数据一致，HPr 对 PRD1 具有负调控作用。这项工作首次揭示了在包含具有不同二聚化特性的重复调节结构域的调节剂中，如何实现特异性，这些结构域由不同的磷酸供体磷酸化控制。此外，结果表明，含有 PRD 的终止抑制蛋白的结构域排列受到选择性压力的影响，以确保适当的调节输出，即在存在相应糖的情况下，靶基因的转录终止抑制作用具有特异性。
