Bergemann K, Nierhaus K H
J Biol Chem. 1983 Dec 25;258(24):15105-13.
In a classical translocation experiment, deacylated RNA is bound to the ribosomal peptidyl-tRNA site (P site) and N-acetyl-phenylalanyl-tRNA (AcPhe-tRNA) to the aminoacyl-tRNA site (A site); upon addition of elongation factor (EF-G) and GTP, AcPhe-tRNA is translocated from the A to the P site. Here, we demonstrate a model reaction for a spontaneous, EF-G independent translocation. If AcPhe-tRNA is bound to the P site and Phe-tRNA to the A site at 15 mM Mg2+, then at 37 degrees C up to 60% of the AcPhe2-tRNA formed is found at the P site without the addition of EF-G. We demonstrate the following: 1) the spontaneous translocation is not merely illusory as a result of Phe-tRNA binding directly to the P site; 2) it is not mimicked by release of AcPhe2-tRNA from an A site and rebinding to a P site of another ribosome; 3) it is not caused by an EF-G contaminant present in the 70 S preparation, since without EF-G the spontaneous translocation works equally well in the presence of guanyl-5'-yl imidodiphosphate or fusidic acid; 4) AcPhe2-tRNA evidently has a higher affinity for the P site than AcPhe-tRNA, thus promoting the spontaneous translocation; and 5) peptide-bond formation favors the subsequent translocation. Addition of EF-G increases the initial rate by a factor of 13. Furthermore, at 15 mM Mg2+, 37 degrees C and in the presence of EF-G and GTP, Phe-tRNA cannot be translocated from the A to the P site, if the P site is occupied by deacylated tRNA. With the spontaneous translocation system, all reactions of the elongation cycle are cooperatively interconnected; i.e. upon binding of aminoacyl-tRNA to the A site, a significant portion of the ribosomes performs a complete round of the elongation cycle without the addition of elongation factor EF-G.
在经典的转位实验中，脱酰基RNA结合到核糖体肽基 - tRNA位点（P位点），而N - 乙酰苯丙氨酰 - tRNA（AcPhe - tRNA）结合到氨酰 - tRNA位点（A位点）；加入延伸因子（EF - G）和GTP后，AcPhe - tRNA从A位点转位到P位点。在此，我们展示了一种自发的、不依赖EF - G的转位模型反应。如果在15 mM Mg2 +条件下，AcPhe - tRNA结合到P位点，苯丙氨酰 - tRNA（Phe - tRNA）结合到A位点，那么在37℃时，即使不添加EF - G，也能发现高达60%新形成的AcPhe2 - tRNA位于P位点。我们证明了以下几点：1）自发转位并非仅仅是由于Phe - tRNA直接结合到P位点而产生的假象；2）它并非由AcPhe2 - tRNA从A位点释放并重新结合到另一个核糖体的P位点所模拟；3）它不是由70 S制剂中存在的EF - G污染物引起的，因为在没有EF - G的情况下，自发转位在鸟苷 - 5'- 亚基亚氨基二磷酸或夫西地酸存在时同样有效；4）AcPhe2 - tRNA对P位点的亲和力显然高于AcPhe - tRNA，从而促进了自发转位；5）肽键形成有利于随后的转位。添加EF - G可使初始速率提高13倍。此外，在15 mM Mg2 +、37℃以及存在EF - G和GTP的情况下，如果P位点被脱酰基tRNA占据，Phe - tRNA就无法从A位点转位到P位点。在自发转位系统中，延伸循环的所有反应都是协同相互关联的；也就是说，当氨酰 - tRNA结合到A位点时，很大一部分核糖体在不添加延伸因子EF - G的情况下就能完成一轮完整的延伸循环。
