Abstract:[Background] Thermophilic group II introns are a class of retrotransposons that are composed of intron RNA and intron-encoded protein (IEP) and can move on chromosomes at high frequency under high temperature, and nowadays, they have been used to develop Themortargetron, an efficient gene-targeting tool. Therefore, it is highly important to elucidate their active catalytic sites for studying the "retrohoming" mechanism and developing new genetic tools. [Objective] To screen the key active sites from the domain of thermophilic group II intron Tel3c/4c-RT and obtain IEP mutants with inactivated reverse transcription function. [Methods] Firstly, bioinformatics method was used to analyze and screen the key amino acid sites that might affect the reverse transcription function of Tel3c/4c-RT. Then, site-directed mutations were performed on the selected key amino acid sites, and Thermotargetron plasmids were used to construct a targeting system of thermophilic group II intron with inactivated reverse transcription function. Finally, taking the lacZ gene of Escherichia coli as example, the targeting efficiency of the Thermotargetron mutant system was analyzed by blue-white screening, and the effect of the key active site of the Tel3c/4c-RT on the targeting efficiency of the thermophilic group II intron was verified in vivo. [Results] A total of 15 amino acid sites that may affect reverse transcriptional activity were screened out: D194, I195, S196, G197, C198, F199, Q241, G242, R274, Y275, A276, D277, D278, L324 and G325. The "retrohoming" of Tel3c/4c was almost completely lost when the G242 and R274 were mutated, suggesting that the G242 and R274 were the core catalytic sites affecting the reverse transcription function of Tel3c/4c-RT. [Conclusion] The core catalytic sites affecting the reverse transcription function of Tel3c/4c-RT were screened out, which laid a good foundation for in-depth study of the "retrohoming" mechanism of the thermophilic group II introns and the further development.
