Cullen P J, Bowman W C, Kranz R G
Department of Biology, Washington University, St. Louis, Missouri 63130, USA.
J Biol Chem. 1996 Mar 15;271(11):6530-6. doi: 10.1074/jbc.271.11.6530.
Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54-independent promoters. To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4-fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc- NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter.
肠道细菌中增强子依赖性转录依赖于一种结合在启动子上游远处DNA的激活蛋白和一种在启动子处与核心RNA聚合酶结合的替代σ因子（σ54）。在光合细菌荚膜红细菌中，NtrB和NtrC蛋白（RcNtrB和RcNtrC）是一个双组分系统的假定成员，该系统很新颖，因为结合增强子的RcNtrC蛋白激活不依赖σ54的启动子的转录。为了在体外重建这个假定的双组分系统，RcNtrB蛋白在大肠杆菌中过表达，并作为麦芽糖结合蛋白融合体（MBP-RcNtrB）进行纯化。MBP-RcNtrB在体外自磷酸化至与鼠伤寒沙门氏菌NtrB（StNtrB）蛋白相同的稳态水平且具有相同的稳定性，但初始速率较低。MBP-RcNtrB在体外使鼠伤寒沙门氏菌NtrC（St-NtrC）和RcNtrC蛋白自磷酸化。肠道NtrC蛋白在体内也被RcNtrB磷酸化，因为编码RcNtrB或MBP-Rc-NtrB的质粒激活NtrC依赖性nifL-lacZ融合体的转录。向RcNtrC的磷酸转移速率和磷酸化的RcNtrC（RcNtrC---P）的自磷酸酶活性与StNtrC蛋白相当。然而，RcNtrC蛋白似乎是一种特异性的RcNtrB P磷酸酶，因为RcNtrC不会被小分子磷酸盐化合物或StNtrB蛋白磷酸化。RcNtrC在溶液中形成二聚体，并且RcNtrC - P与g1nB启动子的上游串联结合位点的结合能力比未磷酸化的RcNtrC蛋白强4倍，这可能是由于RcNtrC -P的寡聚化。因此，荚膜红细菌的NtrB和NtrC蛋白形成了一个类似于其他NtrC样系统的双组分系统，其中RcNtrB向RcNtrC蛋白的特异性磷酸转移导致在增强子处的寡聚化增加，但随后激活不依赖σ54的启动子。
