游仆虫肽链释放因子eRF1对编程性翻译移码的影响  

The Influence of Euplotic Polypeptide Chain Release Factor eRF1 on Programmed Translational Frameshift

作　　者：李禄琴 胡苗清[1] 王软林[1] 柴宝峰[1] 梁爱华[1] 

机构地区：[1]山西大学生物技术所,化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《中国生物化学与分子生物学报》2014年第5期468-474,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31372199;No.31272100);山西省回国留学人员科研项目(2012-1)~~

摘　　要：编程性翻译移码是mRNA翻译为多肽链时核糖体沿mRNA正向或反向滑动1个碱基才能表达出1个完整多肽链的现象.人的肽链释放因子eRF1对HIV-1病毒的编程性-1移码有直接的影响.而且在频繁发生编程性+1移码的单细胞真核生物游仆虫中,肽链释放因子eRF1对编程性移码也有明显的影响.为进一步研究eRF1中影响编程性翻译移码的关键序列及调控机理,本研究将含有不同终止密码子的移码序列和已报道的游仆虫移码基因Ndr2分别插入双荧光素酶报告基因中,成功建立了可在酵母中进行研究的编程性移码报告检测体系.利用游仆虫肽链释放因子Eo-eRF1b的N结构域和酵母肽链释放因子Sc eRF1的MC结构域构建了杂合肽链释放因子(Eo/Sc eRF1),检测Eo-eRF1b N结构域中的不同突变位点对移码效率的影响.结果表明,游仆虫肽链释放因子eRF1b中YCF区的突变能明显促进含终止密码UAA的移码序列的移码,推测这可能是由于eRF1突变体降低了对UAA的识别所导致.此外,杂合肽链释放因子Eo/Sc eRF1能够有效地提高移码基因Ndr2的移码效率.eRF1b中YCF区的突变同样能明显促进Ndr2的移码.因此,游仆虫肽链释放因子YCF区的特殊序列可能是这种生物中发生编程性移码频率较高的原因之一.本研究为探讨纤毛虫编程性翻译移码调控机制提供了实验数据.Programmed translational frameshift is a phenomenon whereby the ribosome is guided to shift one nucleotide position upstream （ ＋ 1 ） or downstream （ - 1 ） to produce the correct peptidyl products. Human peptide chain release factor （eRF1） was known to affects -1 programmed translational frameshift of the HIV-1 virus, as well as to impact the ＋ 1 programmed frameshift frequently occurs in euplotes. To identify the key sequence of eRF1 for its function in programmed frameshift, different sequences containing frameshift stop and the frameshift gene Ndr2 of Euplotes octocarinatus were inserted into a dual luciferase reporter vector, respectively, and tested for translation in yeast. The hybrid peptide chain release factor Eo/Sc eRF1 of the N domain of Euplotes octocarinatus eRF1 b was fused with MC domains of Saccharomyces cerevisiae eRF1, the efficiency of frameshift was measured upon various mutations in the Eo-eRFlb N domain. The results showed that the YCF motif in Eo/Sc eRFlb increased the frameshift at the UAA codon. It suggested that the YCF sequence of eRF1 played an important role in programmed frameshift in euplotes, suggesting its importance in the frameshift of Ndr2. The results provided preliminary data for further investigation about the molecular mechanisms of programming translational frameshift in ciliates.

关 键 词：八肋游仆虫 编程性翻译移码 肽链释放因子 双荧光素酶报告体系 

分 类 号：Q75[生物学—分子生物学]