Meier T I, Peery R B, Jaskunas S R, Zhao G
Infectious Diseases Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285-0438, USA.
J Bacteriol. 1999 Sep;181(17):5242-9. doi: 10.1128/JB.181.17.5242-5249.1999.
Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione S-transferase (GST) fusion forms of Streptococcus pneumoniae Era in Escherichia coli and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of S. pneumoniae during affinity purification and remained associated with the protein after removal of the GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic properties (dissociation constant [kD], Km, and Vmax). However, the native Era protein purified by using different chromatographic columns had a much lower GTPase activity than did GST-Era, although it had a similar k(D). In addition, RNA was not associated with the protein. Purified GST-Era protein was shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a significant reduction in GTPase activity. The RNA associated with GST-Era was shown to be predominantly 16S rRNA. The native Era protein isolated directly from S. pneumoniae was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is associated with Era and might stimulate its GTPase activity.
Era是一种存在于细菌和支原体中的重要膜相关GTP酶。Era似乎在细菌细胞周期调控中发挥重要作用。在本研究中，我们在大肠杆菌中表达了肺炎链球菌Era的天然形式和谷胱甘肽S-转移酶（GST）融合形式，并将两种蛋白均纯化至同质。我们发现，在亲和纯化过程中，RNA与肺炎链球菌的GST-Era蛋白共纯化，并且在通过凝血酶切割去除GST标签后仍与该蛋白结合。经凝血酶处理和未处理的GST-Era蛋白均可结合并水解GTP，且表现出相似的动力学特性（解离常数[kD]、Km和Vmax）。然而，尽管天然Era蛋白与GST-Era具有相似的k(D)，但使用不同色谱柱纯化得到的天然Era蛋白的GTP酶活性要低得多。此外，RNA不与该蛋白结合。纯化后的GST-Era蛋白以高分子量（600 kDa）和低分子量（120 kDa）形式存在。GST-Era的高分子量形式与RNA相关，并表现出非常高的GTP酶活性。约40%的纯化GST-Era蛋白与RNA相关，去除RNA会导致GTP酶活性显著降低。与GST-Era相关的RNA主要为16S rRNA。直接从肺炎链球菌中分离得到的天然Era蛋白也以与RNA复合的高分子量形式（600 kDa）存在。总之，我们的结果表明16S rRNA与Era相关，并可能刺激其GTP酶活性。
