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。 人类基因组项目的完成表明基因很少，但是大量的非蛋白质编码（NC）RNA。例如，仅小鼠基因组中有〜4500个NCRNA，只有少数具有已建立的分子功能。发夹核酶是一种源自血1植物病原体病毒体的NCRNA。发夹核酶的生物学功能是通过在其同源底物中的RNA介导的，特异性的磷酸二酯键裂解反应的作用来裂解串联转录物至单位长度。发夹核酶代表了一个模型系统，用于研究NCRNA结构和功能，这是由于其尺寸较小，易于检测到的催化活性以及其形成良好分裂晶体的能力。 该提案的目的是阐明发夹核酶的功能群，从而有助于其百万倍的加速度。 最近对A38和G8 abasic头蛋白核酶变体的动力学分析表明，天然RNA的催化不会通过使用A38和G8的一般酸/碱机制进行催化（如先前提出的），而是使用过渡态稳定化，这些稳定稳定涉及涉及涉及本质上添加的救助基础贡献的特定静电组的过渡稳定。此外，该反应可能与1-2个水一起起作用，该水作为特定的酸/碱催化剂。 申请人的实验室先前证明，发夹核酶活性位置存在水，与所提出的特定基础机制一致（Salter等人和Wedekind，2006年）。对于当前的提案，我们与过渡态模拟物（钒酸盐）中的复合物中产生了最小的全RNA发夹核酶的晶体，该晶体应揭示高分辨率的其他特异性酸/碱催化剂。 同样，我们制备了一系列G8或A38 abasic发夹核酶的晶体。 所有晶体都已在我们在罗切斯特的家用源上进行了预先筛选。晶体表现出衍射范围为2.8至2.3埃脉冲。 以前，我们证明了在梁线A-1处发夹核酶衍射的明显改善，从而鉴定了碱基U37处的构象异质性，并提出了U39C功能增益的建议（Alam等人＆Wedekind，2005）。我们估计，我们将需要约40小时的衍射数据，这将包括各种具有不同救援基地的无碱性样本。这些结构应揭示在催化态下的救助基础结合的方式，以及在过渡态下假定的催化水的存在。这些观察结果对于回到我们的家庭实验室的理性动力学分析至关重要。最终，结果应通过证明对拟议的水介导的作用机理的结构支持来推动田地前进。 此外，对发夹核酶的更深入的了解将在分析其他NCRNA时提供化学和结构先例。 例如，在50S核糖体肽基转移位点观察到水，但是没有直接的证据表明溶剂在催化中起着至关重要的作用。 最后，我们建议收集有关代表乙型肝炎病毒的TRP/琥珀色编辑位点的43-MER RNA晶体的数据（Macelrevey＆Wedekind，2005）。这些晶体是碘化样品，将用于多种同构置换。 这项研究的目的是阐明蛋白质ADAR1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。 衍生数据集的收集将需要约10小时。