游仆虫编程性核糖体移码的分子机制研究

Programmed ribosomal frameshifting (PRF) is used to regulate gene expression at the translational level. This phenomenon exists in all branches of life. Previous studies have suggested that PRF is common in protozoan Euplotes. However, the frameshift mechanism is still unclear. In this project, Euplotes macronuclear artificial chromosomes containing verified +1 and +2 frameshift genes will be constructed. Potential slippery sites of these genes will be mutated. The relationship of slippery sequence and frameshift type will be confirmed by comparing the green fluorescence protein expression in wild type and mutant; The transcriptional activity of the anticodon stem loop expended tRNALys will be verified. The Lysyl-tRNA synthetase and the dual luciferase reporter vectors will be prepared to construct the in vitro translation examination system. The effect of special tRNALys on +1 programmed ribosomal frameshifting will be studied using the in vitro translation examination system. Based on the data, the regulatory elements involved in programmed ribosomal frameshifting of Euplotes will be clarified and the frameshift mechanism will be revealed. Our research will lay foundation for comparing the frameshift regulation elements in different organisms.

编程性核糖体移码是一种翻译水平上的基因表达调控方式，该现象广泛存在于生命进化的各个分支。原生动物游仆虫中也存在高频率的编程性核糖体移码现象，但其移码机制尚不清楚。本项目拟选取已证实的+1位及+2位移码基因，构建游仆虫绿色荧光蛋白大核人工染色体，并对其滑动序列进行突变，通过比较野生型及突变体中绿色荧光蛋白的表达情况，探讨滑动序列对移码类型的影响；证实前期发现的反密码子环扩大的tRNALys具有转录活性，表达纯化八肋游仆虫赖氨酰tRNA合成酶、构建双荧光素酶报告质粒，建立用于检测移码效率的体外翻译检测体系，利用该体系检测特殊tRNALys对+1位编程性核糖体移码的影响。探讨游仆虫中影响编程性核糖体移码的调控元件，进而阐明其分子机制，为比较不同生物中编程性核糖体移码的调控元件特点奠定基础。

编程性核糖体移码是一种广泛存在于生命进化谱系各个分支的基因表达调控方式。单细胞真核生物游仆虫中有高频率的编程性核糖体移码现象，本项目以八肋游仆虫为材料，从顺式作用原件（滑动序列）及反式作用因子（反密码子环扩大的tRNALys）两方面探讨游仆虫编程性核糖体移码的调控元件及其分子机制。证实了η微管蛋白为+1位移码基因，构建含绿色荧光蛋白报告基因的游仆虫大核人工染色体，转染游仆虫细胞，通过检测GFP的表达来确定不同滑动序列突变体对应的移码类型，证实了滑动序列对游仆虫中识别+1位和+2位移码具有关键作用，且这种移码类型的改变与滑动序列第1个密码子编码何种氨基酸无关；此外，对八肋游仆虫赖氨酰tRNA合成酶进行了异源表达纯化，对tRNALys进行了体外转录，并构建了萤火虫荧光素酶移码报告质粒，建立了可用于检测移码效率的体外翻译检测体系，利用该检测体系明确了反密码子环扩大的特殊tRNALys对+1位编程性核糖体移码无促进作用。研究结果对深入理解编程性核糖体移码的调控元件及分子机制提供了实验数据。