Programmed -1 frameshifting by kinetic partitioning during impeded translocation.
Programmed -1 frameshifting by kinetic partitioning during impeded translocation.
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DOI：
10.1016/j.cell.2014.04.041
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发表时间：
2014-06-19
期刊：
影响因子：
64.5
通讯作者：
Rodnina MV
中科院分区：
文献类型：
--
作者：
Caliskan N;Katunin VI;Belardinelli R;Peske F;Rodnina MV
程序性核糖体移码(−1PRF)是细胞用来增加基因组信息含量和调节基因表达的一种基因重组事件。-1PRF的作用机制及其在翻译伸长过程中的时间尚不清楚。在这里，我们确定了控制-1PRF的步骤，方法是在重组的大肠杆菌体外翻译系统中，跟随核糖体通过IBV 1a/1b基因模型mRNA的移码位点。移码发生在易位的后期，当两个tRNAs与相邻的mRNA滑动序列密码子结合时。该基因下游的假结点影响了30S亚基头部的关闭运动、EF-G的解离以及核糖体tRNA的释放。核糖体滑入-1框架加速了易位的完成，从而进一步有利于在新的阅读框架中进行翻译。在单密码子分辨率下监测-1核糖体移码的动力学。当30s亚基头部的向后移动被阻止时，发生移码。XXXYYYZ mRNA序列上的两个tRNA停滞在嵌合后状态。向-1读框的移位发生在EF-G从核糖体释放之前。这一过程的动力学解构揭示了tRNA与邻近序列密码子的结合以及由此产生的假结损害了30S核糖体的运动，并将其滑到−1位置进入新的翻译框架。
Programmed –1 ribosomal frameshifting (−1PRF) is an mRNA recoding event utilized by cells to enhance the information content of the genome and to regulate gene expression. The mechanism of –1PRF and its timing during translation elongation are unclear. Here, we identified the steps that govern –1PRF by following the stepwise movement of the ribosome through the frameshifting site of a model mRNA derived from the IBV 1a/1b gene in a reconstituted in vitro translation system from Escherichia coli. Frameshifting occurs at a late stage of translocation when the two tRNAs are bound to adjacent slippery sequence codons of the mRNA. The downstream pseudoknot in the mRNA impairs the closing movement of the 30S subunit head, the dissociation of EF-G, and the release of tRNA from the ribosome. The slippage of the ribosome into the –1 frame accelerates the completion of translocation, thereby further favoring translation in the new reading frame. Kinetics of –1 ribosomal frameshifting are monitored at single-codon resolution Frameshifting occurs when the backward movement of the 30S subunit head is impeded Two tRNAs at the XXXYYYZ mRNA sequence are stalled in chimeric POST states The shift to the –1 reading frame occurs prior to EF-G release from the ribosome Programmed ribosomal frameshifting is an mRNA recoding process that allows cells to maximize gene expression. Kinetic deconstruction of this process reveals that the binding of tRNAs to adjacent slippery sequence codons of the mRNA and the resulting pseudoknot impair movement of the 30S ribosome and slip it to the −1 position into a new translational frame.
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