Brown S, Blumenthal T
Proc Natl Acad Sci U S A. 1976 Apr;73(4):1131-5. doi: 10.1073/pnas.73.4.1131.
Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly(C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme. Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results demonstrate that EF-Tu and EF-Ts function as complex in Qbeta replicase and do not perform their known protein biosynthetic function in the RNA synthetic reaction.
大肠杆菌噬菌体Qβ RNA复制酶是一种依赖RNA的RNA聚合酶（依赖RNA的RNA核苷酸转移酶），它是一种四聚体，由一种噬菌体编码的多肽和三种宿主提供的多肽组成，已知这三种宿主提供的多肽在未感染宿主的蛋白质生物合成中发挥作用。其中两种多肽，即蛋白质合成延伸因子EF-Tu和EF-Ts，可以用亚胺二甲酯共价交联形成一种复合物，该复合物缺乏催化单个延伸因子所催化的已知宿主功能的能力。我们使用先前开发的重组系统，研究了交联EF-Tu-Ts复合物对重组复制酶活性的影响。当外源添加的天然EF-Tu-Ts被交联时，复性效率明显高于未交联时。交联的EF-Tu-Ts可以通过其在变性Qβ复制酶复性过程中替代EF-Tu和EF-Ts的能力，从粗制的交联核糖体后上清液中纯化出来。制备了一种用交联的EF-Tu-Ts替代单个延伸因子的Qβ复制酶样品。尽管它缺乏EF-Tu和EF-Ts活性，但它可以正常启动聚（C）和Qβ RNA的转录，并且比对照酶具有大致相同的比活性。发现用交联的EF-Tu-Ts形成的变性Qβ复制酶复性速度比未处理的酶快得多，并且与正常复制酶不同，其复性不受GDP的抑制。结果表明，EF-Tu和EF-Ts在Qβ复制酶中作为复合物发挥作用，并且在RNA合成反应中不执行其已知的蛋白质生物合成功能。
