ARCHAEAL 3'-PHOSPHATE RNA SPLICING LIGASE CHARACTERIZATION

古菌 3-磷酸 RNA 剪接连接酶表征

• 批准号: 8365789
• 负责人: DIETER SOLL
• 金额: $ 1.28万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365789
• 关键词: Active Sites; Animals; Archaea; Archaeal RNA; Binding; Biology; Cells; Cleaved cell; Data; Enzymes; Eukaryota; Excision; Funding; Fungal Genome; Grant; Hydroxyl Radical; Introns; Ligase; Mutation; National Center for Research Resources; Principal Investigator; Protein Family; Proteins; Pyrobaculum; Pyrococcus horikoshii; RNA Ligase (ATP); RNA Splicing; Recombinants; Reporting; Research; Research Infrastructure; Resources; Source; Structure; Transfer RNA; United States National Institutes of Health; animal extract; cost; endonuclease; inorganic phosphate; phosphodiester; plant fungi; protein folding; structural genomics; tRNA Ligase; tRNA Precursor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3'-halves beginning with a 5'-hydroxyl, and 5'-halves ending in a 2',3'-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3'-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2',3'-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3'-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn(2+) binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3'-P RNA ligase.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 从tRNA前体中去除内含子涉及到tRNA剪接内切酶的切割，以产生tRNA3‘-半部以5’-羟基开始，5‘-半部以2’，3‘-环磷酸结束。然后，tRNA连接酶将这种磷酸结合到连接两半的核苷酸间键中。虽然这种3‘-P RNA剪接连接酶活性在近30年前从动物和后来的古生物细胞中的提取液中被检测到，但相关的蛋白质尚未被确定。在这里，我们报道了该连接酶的纯化，并将其归属于仍未确定的RtcB蛋白家族。对重组嗜气性焦杆菌RtcB的研究表明，该酶能够将剪接的tRNA一半连接到成熟大小的tRNA，其中连接的磷酸二酯键包含最初存在于2‘，3’-环磷酸中的磷酸盐。这些数据证实RtcB为古生菌RNA3‘-P连接酶。结构基因组学研究以前得到了一种Horikoshii热球菌RtcB蛋白的晶体结构，其中包含一个新的蛋白质折叠和一个保守的假定的锌(2+)结合裂隙。这一结构指导了我们对嗜氧假单胞菌酶的突变分析。裂解区高度保守的残基(C100A、H205A、H236A)突变使酶失活，提示这些残基是嗜氧假单胞菌连接酶活性部位的一部分。嗜氧假单胞菌连接酶的活性部位与T4 RNA连接酶、植物和真菌的连接酶没有显著的序列相似性。RtcB在古生物和真核生物中的序列保守性表明，真核生物中的RtcB是长期寻找的动物3‘-P RNA连接酶。