原核生物pre-tRNA 5'末端剪切酶的结构和功能研究  

Structural and Functional Studies of the Prokaryotic pre-tRNA 5'End Processing Enzyme

作　　者：邵齐源 李洋洋 张威振 甘建华 SHAO Qiyuan;LI Yangyang;ZHANG Weizhen;GAN Jianhua(School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《复旦学报(自然科学版)》2025年第1期113-120,共8页Journal of Fudan University:Natural Science

摘　　要：5'末端的剪切是pre-tRNA加工和成熟过程中非常重要的一个环节,由RNase P负责催化。不同于常规的RNase P,原核生物中存在一类protein-only RNase P(也称HARP),不需要依赖核酶就能对pre-tRNA的5'末端进行剪切。为了阐明HARP结合和剪切pre-tRNA的分子机制,我们表达和纯化了Thermocrinis ruber来源的HARP蛋白(TrHARP)。定点突变、分子筛和体外活性实验证实TrHARP具有pre-tRNA剪切活性,而且该活性受到蛋白聚合状态的影响。此外,我们还筛选获得了TrHARP蛋白质的晶体,采集了衍射数据并建立了初始的结构模型。尽管TrHARP与Aquifex aeolicus来源的HARP Aq_880的折叠方式类似,但TrHARP与Aq_880在结构中的聚合方式和相对方位存在显著的差异。分子筛以及负染电镜实验结果均显示pre-tRNA的引入会导致HARP结构的解聚,这为进一步阐明HARP结合和催化pre-tRNA的5'末端成熟的分子机制奠定了基础。Precursor tRNA processing is very important for tRNA maturation,in which RNase P is mainly responsible for shearing of the 5'-end.In prokaryotes,there exists a class of RNase P that does not require ribozyme to cleave the 5'-end of pre-tRNA,which is called HARP.To reveal the detailed mechanism for pre-tRNA binding and cleavage by HARP,we expressed and purified HARP from Thermocrinis ruber,and verified its in vitro cleavage activity.Via mutagenesis and in vitro assays,we showed that the oligomization state of Tr HARP is importance for its biological function.We also solved one Tr HARP crystal structure with a resolution of 4.58.Structural comparison showed that the overall folding of Tr HARP is different from that of homologous protein Aq_880.In combination with size-exclusion and negative stain experimental results,our studies suggest that introducing of pre-tRNA will lead to the disassembly of HARP and shade light on pre-tRNA binding mechanism of HARP proteins.

关 键 词：protein-only RNase P pre-tRNA HARP 晶体结构 功能 蛋白表达 

分 类 号：Q518.2[生物学—生物化学]