The ''allosteric three-site model'' of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli
The ''allosteric three-site model'' of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli
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DOI：
10.1073/pnas.93.22.12183
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发表时间：
1996-10-29
影响因子：
11.1
通讯作者：
Wintermeyer, W
中科院分区：
文献类型：
--
作者：
Semenkov, YP;Rodnina, MV;Wintermeyer, W
对于核糖体tRNA出口(E)位点的功能作用，提出了两种不同的模型，有人认为，离开肽基(P)位点的tRNA的瞬时E位点结合通过降低tRNA释放的能量屏障促进延伸因子G （EP-G）依赖的易位[Lill， R., Robertson， J. M. & Wintermeyer， W. (1989) EMBO J. 8,3933 -3938]，另一种“变弹性三位点模型”[Nierhaus， K. H. (1990) Biochemistry 29,4997 -5008]具有稳定，密码子依赖的tRNA结合到E位点，并假设E和氨基酰基(a)位点之间存在偶联，以反合作的方式调节这两个位点的tRNA结合亲和力。扩展了我们对两种相互冲突的模型的测试，我们用异聚mRNA编程的完全活性核糖体进行了易位实验。结果证实，从P位点释放的脱酰基tRNA以动力学不稳定的方式与E位点结合，并且Mg2+ Or多胺增加了结合的亲和力，即E位点的占用。在易位后复合体中E位点占用率高的情况下，用氨基酰基trna填充E位点对E位点没有影响，即，如果通过去除EF-G来避免第二轮易位，则两个位点之间没有可检测到的反合作偶联。在这些结果的基础上，这与我们之前的结果完全一致，我们认为变构的三位点延伸模型是站站的，相反，正如之前提出的那样，离开tRNA的E位点结合状态是一个短暂的中间状态，因此，是经典的延长核糖体两态模型的一个机制特征。
For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed, It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EP-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938], The alternative ''allosteric three-site model'' [Nierhaus, K. H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ Or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the E site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable, Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.