ABASIC RESCUE & THE ROLE OF CAT WATERS IN HAIRPIN RIBOZYME MECHANISM OF ACTION

基本救援

• 批准号: 8363520
• 负责人: Joseph E Wedekind
• 金额: $ 1.64万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363520
• 关键词: ADAR1; Acceleration; Acids; Active Sites; Amber; Back; Binding; Biological Models; Biological Process; Catalysis; Chemical Structure; Cleaved cell; Code; Collection; Complex; Data; Data Set; Electrostatics; Exhibits; Felis catus; Funding; Genes; Goals; Grant; Hepatitis Delta Virus; Heterogeneity; Home environment; Human Genome Project; Kinetics; Length; Life Cycle Stages; Mediating; Molecular; National Center for Research Resources; Plants; Play; Principal Investigator; Proteins; RNA; RNA Editing; Reaction; Research; Research Infrastructure; Resolution; Resources; Ribosomes; Role; Sampling; Series; Site; Solvents; Source; Structure; Transcript; United States National Institutes of Health; Vanadates; Variant; Viral; Virion; Water; base; beamline; catalyst; cost; functional group; gain of function; hairpin ribozyme; mouse genome; pathogen; phosphodiester

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. Completion of the human genome project revealed a paucity of genes, but an unexpectedly large number of non-protein-coding (nc)RNAs. For example, there are ~4500 ncRNAs in the mouse genome alone and only a handful have an established molecular function. The hairpin ribozyme is an ncRNA derived from the virions of sub-viral plant pathogens. The biological function of the hairpin ribozyme is to cleave concatenated transcripts to unit length by action of an RNA-mediated, site-specific phosphodiester bond cleavage reaction within its cognate substrate. The hairpin ribozyme represents a model system to study ncRNA structure and function due to its small size, readily detectable catalytic activity and its ability to form well-diffracting crystals. The goal of this proposal is to elucidate the functional groups of the hairpin ribozyme that contribute to its million-fold rate acceleration. Recent kinetic analyses of A38 and G8 abasic hairpin ribozyme variants suggested that catalysis of the native RNA does not proceed through a general acid/base mechanism using A38 and G8 (as previously proposed), but instead uses transition-state stabilization involving electrostatic contributions from specific function groups contributed by the exogenously added rescue bases. In addition, the reaction may function in concert with 1-2 waters that serve as specific acid/base catalysts. The applicant's lab previously demonstrated that waters were present in the hairpin ribozyme active site, consistent with the proposed specific base mechanism (Salter et al. & Wedekind, 2006). For the current proposal, we have generated crystals of a minimal all-RNA hairpin ribozyme in complex with a transition-state mimic (vanadate) that should reveal additional, putative specific acid/base catalysts at high resolution. Similarly, we have prepared crystals of a series of G8 or A38 abasic hairpin ribozymes. All crystals have been pre-screened on our home source in Rochester. Crystals exhibited diffraction ranging from 2.8 to 2.3 Angstroms resolution. Previously we demonstrated marked improvement of hairpin ribozyme diffraction at beamline A-1, leading to the identification of conformational heterogeneity at base U37 and a proposal of U39C gain of function (Alam et al. & Wedekind, 2005). We estimate that we will require ~40 hrs to collect diffraction data, which will encompass a variety of abasic samples with different rescue bases. These structures should reveal the mode of rescue base binding in the pre-catalytic state, as well as the presence of putative catalytic waters in the transition state. These observations will be essential for rational kinetic analysis back in our home lab. Ultimately, the result should drive the field forward by proving structural support for the proposed water-mediated mechanism of action. In addition, a greater understanding of the hairpin ribozyme will provide chemical and structure precedents in the analysis of other ncRNAs. For example, water has been observed in the 50S ribosome peptidyl transfer site, but there is no direct evidence that solvent plays an essential role in catalysis. Finally, we propose to collect data on RNA crystals of a 43-mer that represents the Trp/Amber editing site of the hepatitis-delta-virus (MacElrevey & Wedekind, 2005). These crystals are iodinated samples that will be used for multiple isomorphous replacement. The goal of this study is to elucidate the structural features leading to the selection of the RNA editing site by the protein ADAR1, which is essential to complete the viral life cycle. Collection of derivative data sets will require ~10 hrs.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 人类基因组计划的完成揭示了基因的缺乏，但非蛋白质编码 (nc)RNA 的数量却出人意料地多。例如，仅小鼠基因组中就有约 4500 个 ncRNA，其中只有少数具有已确定的分子功能。发夹核酶是一种源自亚病毒植物病原体病毒粒子的 ncRNA。发夹核酶的生物学功能是通过其同源底物内RNA介导的、位点特异性磷酸二酯键裂解反应的作用，将串联转录物裂解成单位长度。发夹核酶因其体积小、易于检测的催化活性以及形成良好衍射晶体的能力而成为研究 ncRNA 结构和功能的模型系统。 该提案的目标是阐明发夹核酶的功能组，这些功能组有助于其百万倍的速率加速。 最近对 A38 和 G8 无碱基发夹核酶变体的动力学分析表明，天然 RNA 的催化并不是通过使用 A38 和 G8 的一般酸/碱机制（如之前提出的）进行的，而是使用涉及特定静电贡献的过渡态稳定。外源增加的救援基地所贡献的功能群。此外，该反应可以与用作特定酸/碱催化剂的1-2种水协同作用。 申请人的实验室先前证明发夹核酶活性位点中存在水，这与所提出的特定碱基机制一致（Salter等人和Wedekind，2006）。对于当前的提案，我们已经生成了一种最小的全 RNA 发夹核酶与过渡态模拟物（钒酸盐）复合物的晶体，该晶体应该以高分辨率揭示额外的、假定的特定酸/碱催化剂。 类似地，我们制备了一系列G8或A38脱碱基发夹核酶的晶体。 所有晶体均已在我们位于罗切斯特的家乡进行了预筛选。晶体的衍射分辨率范围为 2.8 至 2.3 埃。 此前，我们证明了光束线 A-1 处发夹核酶衍射的显着改善，导致 U37 碱基构象异质性的鉴定以及 U39C 功能增益的提议（Alam 等人和 Wedekind，2005）。我们估计需要大约 40 小时来收集衍射数据，其中将包含具有不同救援基地的各种无碱样品。这些结构应该揭示预催化状态下救援碱基结合的模式，以及过渡状态下假定的催化水的存在。这些观察结果对于我们家庭实验室的理性动力学分析至关重要。最终，结果应该通过证明所提出的水介导作用机制的结构支持来推动该领域的发展。 此外，对发夹核酶的更深入了解将为其他 ncRNA 的分析提供化学和结构先例。 例如，在50S核糖体肽基转移位点中观察到水，但没有直接证据表明溶剂在催化中发挥重要作用。 最后，我们建议收集 43 聚体 RNA 晶体的数据，该晶体代表丁型肝炎病毒的 Trp/Amber 编辑位点（MacElrevey & Wedekind，2005）。这些晶体是碘化样品，将用于多重同晶置换。 本研究的目的是阐明导致 ADAR1 蛋白选择 RNA 编辑位点的结构特征，这对于完成病毒生命周期至关重要。 收集衍生数据集大约需要 10 小时。