Ribosomal pausing at a frameshifter RNA pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency
Ribosomal pausing at a frameshifter RNA pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency
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DOI：
10.1128/mcb.21.24.8657-8670.2001
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发表时间：
2001-12-01
影响因子：
5.3
通讯作者：
Brierley, I
中科院分区：
文献类型：
--
作者：
Kontos, H;Napthine, S;Brierley, I
在这里，我们使用平移延伸和核糖体足印测定研究了程序性-1核糖体移码位点处的核糖体暂停。确定了传染性支气管炎病毒 (IBV) 移码信号处的暂停位点，该位点与 RNA 假结诱导的暂停一致，该暂停将核糖体 P 和 A 位点置于滑序列上。类似地，在猿猴逆转录病毒 I gag/pol 信号处暂停，该信号包含不同类型的移码器假结，也将核糖体放置在滑序列上，支持停顿在移码中的作用。然而，暂停和移码之间缺乏简单的相关性。首先，与HIV假结密切相关的茎环结构，虽然无法刺激有效的移码，但与亲代假结在mRNA上的相同位置和程度相似地暂停核糖体。其次，仅由单个环2核苷酸不同但在移码中具有不同功能的两个假结诱导了相同的暂停模式。最终的观察结果来自于对阅读阶段对暂停的影响的评估。鉴于核糖体以三联体方式前进，我们测试了核糖体遇到 RNA 结构的阅读框（阅读阶段）是否会影响暂停。我们发现阅读阶段确实影响暂停，但出乎意料的是，在暂停最少的阶段中带有假结的 mRNA 被发现比其他变体更有效地促进移码。总的来说，这些实验支持这样的观点：仅暂停不足以介导移码，需要额外的事件。暂停的相位依赖性可能表明核糖体中的活性需要与 mRNA 二级结构的最佳接触才能有效解旋。
Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus I gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the HIV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.