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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 从tRNA前体中的内含子去除涉及通过tRNA剪接内切核酸酶裂解，以从5'-羟基开始产生3 halves的tRNA，而5'-halves则以2'，3'-循环磷酸盐结尾。然后，tRNA连接酶将这种磷酸盐掺入与两半结合的核苷酸键中。尽管将这种3'-P RNA剪接连接酶活性在大约三十年前在动物和后来的古细胞提取物中检测到，但尚未鉴定出负责的蛋白质。在这里，我们报告了该连接酶从甲虫Kandleri细胞中纯化的纯化，及其分配到仍然未表征的RTCB蛋白家族中。对重组拟金吡咯座RTCB的研究表明，该酶能够将剪接的tRNA半结合到成熟的TRNA，其中连接的磷酸二酯链接包含最初存在于2'，3'-固定磷酸盐中的磷酸盐。数据证实RTCB为古细菌RNA 3'-P连接酶。结构性基因组学的努力先前产生了含有新蛋白质折叠和保守的推定锌（2+）结合裂口的horikosococus horikosococcus horikosococcus rtcb蛋白的晶体结构。该结构指导了我们对气孢菌假单胞菌的突变分析。高度保守的残基突变（C100A，H205A，H236A）使酶不活跃，这表明这些残基是aerophilum P. p. aerophilum rigase的活性位点的一部分。植物和T4 RNA连接酶的活性位点之间没有明显的序列相似性，也没有植物和真菌的连接酶。古细菌和真核生物中的RTCB序列保守性将真核生物RTCB视为长期追求的动物3'-P RNA连接酶。