新冠病毒假结在大肠杆菌中的可调节性移码  

The Regulatory Frameshift of SARS-CoV-2 RNA Pseudoknot in Escherichia coli

作　　者：卜明月 宋青[1] BU Mingyue;SONG Qing(School of Chemistry and Biological Engineering,University of Science and Technology of Beijing,Beijing 100083,China)

机构地区：[1]北京科技大学化学与生物工程学院,北京100083

出　　处：《中国生物工程杂志》2025年第1期1-12,共12页China Biotechnology

基　　金：北京科技大学大学生国际竞赛资助项目。

摘　　要：目的:新型冠状病毒(SARS-CoV-2)在宿主细胞内连续翻译病毒mRNA时可以通过程序性移码实现分阶段表达其功能蛋白,移码主要依赖基因组上一段特殊的RNA折叠结构假结(pseudoknot);假结序列在冠状病毒中高度保守,与滑动位点和连接区组成移码刺激元件(frameshift stimulation element,FSE);已有研究报道冠状病毒FSE在原核系统仍然可引起移码翻译,其有效长度仅需88个核苷酸(nucleotides,nt);FSE与相邻序列可互作形成多种二级结构,所以选取新冠FSE序列88 nt及其随后49 nt序列为研究对象,探究加长的新冠FSE(对应氨基酸序列称假结肽)在原核核糖体上移码翻译的效率以及移码翻译的条件依赖性。方法:通过构建由大肠杆菌表达编码红色荧光蛋白(red fluorescent protein,RFP)-假结肽-半乳糖苷酶α片段(lacZ')三联蛋白的嵌合基因,可以实现RFP与lacZ'由假结肽连接的串联表达,从而根据菌落颜色变化来指示FSE介导的移码翻译。结果:实验结果显示,菌落中嵌合蛋白表达呈不均匀状,特别是在荧光显微镜下观察时,RFP荧光信号分布差异巨大;SDS-PAGE蛋白电泳证实新冠FSE在大肠杆菌表达体系可以产生移码翻译。结论:通过构建多种新冠FSE突变体,验证了移码滑动序列以及连接区长度的重要性;进一步发现新冠FSE在原核系统中也存在非移码翻译与移码翻译的动态平衡,其发生受菌落密度、培养温度,甚至低浓度盐酸胍的影响;最后,使用该嵌合基因表达平台筛选出庆大霉素修饰物,该修饰物作为外源小分子影响新冠假结FSE移码效率。Objective:SARS-CoV-2 is capable of expressing its functional proteins in a stage-separated manner within host cells,relying primarily on its unique RNA folding structures,which induce a frameshift during translation of viral genes on host ribosomes.This highly conserved region forms unique secondary structures known as pseudoknots,along with slippery sequence,and connecting nucleotides(nt),forming a so-called frameshift stimulation element(FSE).FSE introduces an invariant programmed-1 ribosomal frameshifting(-1 PRF)at a specific slippery site.Previous studies have demonstrated that even in prokaryotic systems,the coronavirus FSE structure of only 88 nt can induce frameshift expression.Given that multiple secondary structural variations exist for the FSE and neighboring region,88 nt do not encompass all potential secondary structures,the 88 nt FSE together with its following 49 nt sequence is selected to investigate frameshift phenomena.Methods:The current study aimed to explore frameshift translation of an extended SARS-CoV-2 FSE(translated as pseudoknot peptide)in Escherichia coli and its influencing factors.A fusion reporter gene was constructed for E.coli expression,encoding red fluorescent protein(RFP),followed by a pseudoknot peptide and a lacZ'fragment(theαsubunit ofβ-galactosidase).Simultaneous expression of RFP and lacZ'allows detection of frameshift translation events by observing changes in colony color.Results:Fluorescence microscopy results demonstrated strikingly uneven patterns of fusion gene expression across colonies.SDS-PAGE analyses reveal that frameshift translation does occur through SARS-CoV-2's FSE on E.coli ribosomes,but frameshift efficiency remains very low compared to efficiency in the human host.Conclusion:By generating various mutants of FSE,the key slippery sequence and the importance of linker length essential for frameshift are confirmed.Further studies reveal that SARS-CoV-2's FSE maintains a dynamic balance between non-frameshift and frameshift translation in E.coli,which is inf

关 键 词：RNA假结 程序性移码 SARS-CoV-2 大肠杆菌 红色荧光蛋白 错误折叠 

分 类 号：Q78[生物学—分子生物学]