Xin H, Leanza K, Spremulli L L
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290, USA.
Biochim Biophys Acta. 1997 May 2;1352(1):102-12. doi: 10.1016/s0167-4781(97)00003-1.
When bovine mitochondrial elongation factor Ts (EF-Ts(mt)) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli EF-Tu (EF-Tu(Eco) x Ts(mt)). This complex is active in poly(U)-directed polymerization and this activity is inhibited by kirromycin. The EF-Tu(Eco) x Ts(mt) complex does not bind guanine nucleotides detectably and is not dissociated to a significant extent by either GDP or GTP. A portion of the EF-Tu(Eco) x Ts(mt) complex can be dissociated by aa-tRNA in the presence of GTP. The heterologous complex cannot be dissociated completely in the presence of either the 8 M urea or 8 M guanidine hydrochloride, suggesting that EF-Ts(mt) has an unusually tight interaction with E. coli EF-Tu. The EF-Tu(Eco) x Ts(mt) complex can be dissociated by denaturation using 2 M guanidine thiocyanate. Free EF-Ts(mt) can then be purified and renatured. The refolded EF-Ts(mt) is active in stimulating the activity of expressed mitochondrial EF-Tu (EF-Tu(mt)) in poly(U)-directed polymerization. Almost all the EF-Ts(mt) molecules appear to refold into a conformation which can interact with EF-Tu(mt). Protease mapping of EF-Ts(mt) indicates that the first 54 residues fold into an independent domain. Analysis of deletion derivatives of EF-Ts(mt) indicates that extensive regions of this factor are required for its tight interaction with EF-Tu.
当牛线粒体延伸因子Ts（EF-Ts(mt)）在大肠杆菌中表达时，它会与大肠杆菌EF-Tu形成紧密结合的复合物（EF-Tu(Eco)×Ts(mt)）。该复合物在聚尿苷酸（poly(U)）指导的聚合反应中具有活性，且这种活性受奇霉素抑制。EF-Tu(Eco)×Ts(mt)复合物无法检测到与鸟嘌呤核苷酸结合，并且在很大程度上不会被GDP或GTP解离。在GTP存在的情况下，一部分EF-Tu(Eco)×Ts(mt)复合物可被氨酰-tRNA解离。在8M尿素或8M盐酸胍存在时，这种异源复合物都不能完全解离，这表明EF-Ts(mt)与大肠杆菌EF-Tu的相互作用异常紧密。EF-Tu(Eco)×Ts(mt)复合物可用2M硫氰酸胍变性解离。然后可纯化并复性游离的EF-Ts(mt)。复性后的EF-Ts(mt)在聚尿苷酸指导的聚合反应中能刺激表达的线粒体EF-Tu（EF-Tu(mt)）的活性。几乎所有的EF-Ts(mt)分子似乎都能复性成一种可与EF-Tu(mt)相互作用的构象。对EF-Ts(mt)进行蛋白酶图谱分析表明，其前54个残基折叠成一个独立结构域。对EF-Ts(mt)缺失衍生物的分析表明，该因子的广泛区域对于其与EF-Tu的紧密相互作用是必需的。
