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

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。 人类基因组计划的完成揭示了基因的匮乏，但出人意料的是大量的非蛋白质编码(NC)RNA。例如，仅在小鼠基因组中就有大约4500个ncRNA，只有少数具有既定的分子功能。发夹状核酶是一种来源于亚病毒植物病原体病毒粒子的核糖核糖核酸。发夹状核酶的生物学功能是通过RNA介导的同源底物中位置特异性的磷酸二酯键断裂反应将串联的转录本切割成单位长度。发夹状核酶是研究ncRNA结构和功能的模型系统，因为它体积小，催化活性容易检测，并且能够形成良好的衍射性晶体。 这项建议的目标是阐明发夹核酶的官能团，这些官能团有助于其百万倍的速度加速。最近对A38和G8基本发夹状核酶变体的动力学分析表明，天然RNA的催化不是通过使用A38和G8(如先前所建议的)的一般酸碱机制进行的，而是使用过渡态稳定，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，该反应可能与作为特定酸/碱催化剂的1-2水协同工作。 申请人的实验室以前证明发夹核酶活性部位中存在水，这与提出的特定碱基机制一致(Salter等人)。&Wedekind，2006)。对于目前的提议，我们已经生成了一个最小的全RNA发夹状核酶的晶体与过渡态模拟(钒酸)，这应该会揭示额外的，假定特定的酸/碱催化剂在高分辨率。同样，我们已经制备了一系列G8或A38基本发夹状核酶的晶体。所有的水晶都已经在我们位于罗切斯特的家中进行了预筛选。晶体的衍射分辨率从2.8埃到2.3埃不等。先前，我们证明了在光束线A-1处发夹状核酶衍射的显著改善，从而鉴定了U37碱基的构象异质性，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大约40小时来收集衍射数据，这将包括具有不同救援基地的各种基本样品。这些结构应该揭示前催化状态下的救援碱基结合模式，以及过渡状态下假定的催化水的存在。这些观测对于我们在自己的实验室进行合理的动力学分析是必不可少的。最终，结果应该会通过证明拟议的水中介作用机制得到结构性支持，从而推动该领域向前发展。此外，对发夹状核酶的更好理解将为其他ncRNAs的分析提供化学和结构先例。例如，已经在50s核糖体肽转移位置观察到水，但没有直接证据表明溶剂在催化中起重要作用。 最后，我们建议收集关于43-聚体的RNA晶体的数据，该数据代表了丁型肝炎病毒的Trp/Amber编辑位点(MacElrevey&Wedekind，2005)。这些晶体是碘化的样品，将用于多次同象替换。本研究的目的是阐明导致蛋白质ADAR1选择RNA编辑位点的结构特征，ADAR1是完成病毒生命周期所必需的。收集派生数据集将需要大约10小时。

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.