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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 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。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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的数量出乎意料地多。例如，仅在小鼠基因组中就有约4500个ncRNA，只有少数具有确定的分子功能。发夹状核酶是一种来源于植物亚病毒病原体病毒粒子的ncRNA。发夹核酶的生物学功能是通过RNA介导的位点特异性磷酸二酯键切割反应在其同源底物内将串联的转录物切割成单位长度。发夹状核酶是研究ncRNA结构和功能的模型系统，因为它的小尺寸，易于检测的催化活性和形成衍射良好的晶体的能力。 这个提议的目标是阐明发夹状核酶的功能基团，这些功能基团有助于其百万倍的速率加速。 A38和G8无碱基发夹核酶变体的最近动力学分析表明，天然RNA的催化不通过使用A38和G8的一般酸/碱机制进行（如先前提出的），而是使用过渡态稳定化，涉及由外源添加的救援碱基贡献的特定官能团的静电贡献。此外，反应可与1-2种用作特定酸/碱催化剂的沃茨协同作用。 申请人的实验室先前证明了沃茨存在于发夹核酶活性位点中，与所提出的特异性碱基机制一致（Salter等人和Wedekind，2006）。对于目前的建议，我们已经产生了一个最小的所有RNA发夹核酶的晶体与过渡态模拟（钒酸盐），应该揭示额外的，推定的特定酸/碱催化剂在高分辨率的复杂。 类似地，我们已经制备了一系列G8或A38无碱基发夹核酶的晶体。 所有的水晶都已经在我们罗切斯特的线人那里预先筛选过了。晶体显示出2.8至2.3埃分辨率的衍射。 之前，我们证明了发夹核酶在光束线A-I处衍射的显著改善，导致在碱基U37处构象异质性的鉴定和U39 C功能获得的提议（Alam等人和Wedekind，2005）。我们估计我们将需要约40小时来收集衍射数据，这将包括具有不同救援碱基的各种无碱基样品。这些结构应该揭示了在预催化状态下的救援碱基结合模式，以及在过渡状态下推定的催化沃茨的存在。这些观察结果对于我们实验室的合理动力学分析是必不可少的。最终，研究结果应通过为拟议的水介导的行动机制提供结构性支持，推动该领域向前发展。 此外，对发夹状核酶的进一步了解将为其他ncRNA的分析提供化学和结构先例。 例如，在50 S核糖体肽基转移位点观察到水，但没有直接证据表明溶剂在催化中起重要作用。 最后，我们建议收集43聚体RNA晶体的数据，该晶体代表肝炎-δ-病毒的Trp/Amber编辑位点（MacElrevey & Wedekind，2005）。这些晶体是碘化样品，将用于多个同晶置换。 本研究的目的是阐明导致蛋白质ADAR 1选择RNA编辑位点的结构特征，这对完成病毒生命周期至关重要。 收集衍生数据集将需要约10小时。