基于SHAPE-seq技术解析大肠杆菌典型5′mRNA结构特征及其对基因表达的影响

SHAPE-seq reveals the typical 5′mRNA structure of Escherichia coli and its effect on gene expression

摘要:

【目的】 在原核生物基因表达过程中，由于转录本mRNA半衰期较短，其抗降解能力和招募核糖体启动翻译反应的能力对基因表达的影响显著。然而mRNA在其5′端存在多个功能区域，可以影响其半衰期的长短进而影响目的基因的表达，其中主要包括：Shine-Dalgarno (SD)序列以及其上下游的核糖体“等候”位点(translational standby site, TSS)、N端部分编码序列(N-terminal coding sequence, NCS)。各区域由于其自身和跨区域的结构差异会影响基因表达，因此解析各功能区域的构效关系至关重要。【方法】 采用引物延伸测序(SHAPE-seq)技术，以大肠杆菌为宿主解析了7种结构不同的5′mRNA胞内的结构特点，采集了其调控下mRNA丰度以及蛋白表达量。【结果】 发现非结构化的NCS调控下mRNA丰度和蛋白量分别提高了10倍、19倍；形成二级结构茎长为10 nt的TSS有利于提高mRNA丰度；SD序列被包裹形成二级结构时会影响其介导的翻译起始效率(蛋白表达下降10%)；上述较优的TSS和NCS的组合调控下，mRNA丰度和蛋白量显著提高(11倍、60倍)。【结论】 本研究解析了5′mRNA各区域有利于原核生物基因表达的结构特征，初步建立了各功能区域的构效关系，为工业微生物目标基因表达提供了新型调控元件。

关键词:核糖体等候位点;N端编码序列;SD序列;引物延伸测序;构效关系

Abstract:

[Objective] Due to the short half-life of mRNA transcript, their anti-degradation ability and ability to recruit ribosomes to initiate translation have significant effects on the gene expression in prokaryotes. However, the multiple functional regions in the 5′ end, of mRNA can affect the half-life and thus the expression of target genes, including the Shine-Dalgarno (SD) sequence and its upstream and downstream translational standby sites (TSSs) and the N-terminal coding sequence (NCS). The own and cross-regional structural differences will affect gene expression. Therefore, it is important to analyze the structure-activity relationship of each functional region. [Methods] We employed primer extension sequencing (SHAPE-seq) to analyze the structural characteristics of seven different 5′mRNAs in Escherichia coli and collected the mRNA abundance and protein level information under their regulation. [Results] Under the regulation of unstructured NCS, the mRNA abundance and protein level were increased by 10 and 19 times, respectively. The formation of secondary structure TSS with a stem length of 10 nt increased the mRNA abundance. When the SD sequence was wrapped to form a secondary structure, the efficiency of translation initiation mediated by the SD sequence was affected (protein level downregulation by 10%). The combination of TSS and NCS significantly increased the mRNA abundance and protein level by 11 and 60 folds, respectively. [Conclusion] We characterized the structure of each region of 5′mRNA conducive to prokaryotic gene expression and revealed the structure-activity relationship of each functional region, providing a new regulatory element for target gene expression in industrial microorganisms.

Key words:translational standby site;N-terminal coding sequence;SD sequence;SHAPE-seq;structure-activity relationship