L1.LtrB内含子编码蛋白反转录结构域关键催化位点分析及功能验证

Key catalytic sites in the reverse transcription domain of Ll.LtrB intron encoded protein

摘要:

[目的] 筛选影响Ll.LtrB内含子编码蛋白（Intron encoded protein，IEP）反转录功能的关键催化位点，并获得无反转录活性的IEP突变体。[方法] 首先，利用NCBI数据库，通过序列比对及同源建模方法筛选影响IEP反转录功能的关键氨基酸催化位点；然后，对筛选获得的关键催化位点进行定点突变，同时以Targetron载体为模板，构建无反转录功能的突变型Targetron打靶系统；最后，以大肠杆菌lacZ基因为例，体内验证IEP突变体的功能及其对II型内含子"归巢"效率的影响。[结果] 筛选到C164和G214两个位点是影响内含子编码蛋白反转录功能的关键氨基酸残基，并获得C164K和G214W两个突变体。体内功能分析表明，此两个位点突变完全失活了II型内含子的"归巢"功能。[结论] 筛选并获得了失活反转录功能的Ll.LtrB内含子编码蛋白突变体，为深入研究II型内含子的结构和"归巢"机理奠定了基础。

Abstract:

[Objective] To screen the key catalytic sites that affect the reverse transcription function of intron-encoded protein (IEP) from Ll.LtrB, and to obtain the IEP mutant without reverse transcription activity. [Methods] The key catalytic sites affecting the reverse transcription of IEP were screened by sequence alignment and homology modeling methods. Then, the screened key catalytic sites were subjected to site-directed mutagenesis. The mutated IEP was combined with the Targetron vector to construct the mutated Targetron targeting system without reverse transcription function. Finally, the function of mutant IEP was verified by using the lacZ gene in Escherichia coli.[Results] The sites C164 and G214 of IEP were screened and mutated, completely inactivated the "retrohoming" function in vivo. [Conclusion] The IEP mutants of Ll.LtrB obtained in our study laid a solid foundation for further research on the structure and mechanism of group II intron.