Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences.
Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences.
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DOI：
10.1093/nar/gkx061
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发表时间：
2017-05-19
影响因子：
14.9
通讯作者：
Salis HM
中科院分区：
文献类型：
--
作者：
Espah Borujeni A;Cetnar D;Farasat I;Smith A;Lundgren N;Salis HM
mRNA的翻译速率由几个序列决定因素控制，包括其编码序列n端区域内RNA结构的存在。然而，控制这种mRNA结构何时抑制翻译的物理规则仍不清楚。在这里，我们将系统设计的RNA发夹引入到报告蛋白的n端编码区，与起始密码子的距离稳步增加，然后表征它们在大肠杆菌中的mRNA和表达水平。我们发现，当mRNA结构与核糖体的足迹重叠时，mRNA的翻译率被抑制了多达530倍。相比之下，当mRNA结构位于核糖体足迹之外时，翻译被抑制了<2倍。通过将我们的测量结果与生物物理模型相结合，我们确定核糖体足迹将13个核苷酸延伸到n端编码区，当mRNA结构与核糖体足迹重叠或部分重叠时，仅展开重叠结构的自由能控制翻译抑制的程度。总的来说，我们的研究结果提供了n端编码区翻译起始规则的精确量化，改进了调节翻译速率的转录后调控元件的预测设计。
A mRNA's translation rate is controlled by several sequence determinants, including the presence of RNA structures within the N-terminal regions of its coding sequences. However, the physical rules that govern when such mRNA structures will inhibit translation remain unclear. Here, we introduced systematically designed RNA hairpins into the N-terminal coding region of a reporter protein with steadily increasing distances from the start codon, followed by characterization of their mRNA and expression levels in Escherichia coli. We found that the mRNAs’ translation rates were repressed, by up to 530-fold, when mRNA structures overlapped with the ribosome's footprint. In contrast, when the mRNA structure was located outside the ribosome's footprint, translation was repressed by <2-fold. By combining our measurements with biophysical modeling, we determined that the ribosomal footprint extends 13 nucleotides into the N-terminal coding region and, when a mRNA structure overlaps or partially overlaps with the ribosomal footprint, the free energy to unfold only the overlapping structure controlled the extent of translation repression. Overall, our results provide precise quantification of the rules governing translation initiation at N-terminal coding regions, improving the predictive design of post-transcriptional regulatory elements that regulate translation rate.