THE HAIRPIN RIBOZYME: STRUCTURE, MUTAGENESIS AND MECHANISM OF ACTION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。发夹状核酶是一种催化RNA，属于小核酶家族。这类酶的成员执行特定部位的切割反应，包括剪切性磷的亲核攻击，导致形成环状2？，3？-磷酸二酯和自由的5‘-羟基。在小核酶中，发夹是不寻常的，因为它不需要二价金属离子来催化。最近的结构和化学研究表明，反应通过一般的碱/酸催化机理进行，包括保守的碱基G8和A38。有助于氧磷烷过渡态静电稳定的残基也是必需的，但尚未完全确定。这项建议的目的是研究发夹状核酶在G8总碱基位置和剪切键(A-1)的几个突变变体的结构。申请人S实验室已经以61聚体合成全核糖核酸结构的形式结晶了这些突变体，该结构以前在光束线A-1(国际象棋)上衍射到2.19埃分辨率。这些结果导致：(I)任何自然存在的核酶的最高分辨率全核糖核酸结构，(Ii)鉴定了亲核位点上的惰性2？O-甲基，以及(Iii)在S-TURN结构基序中定位了稳定的CoHexaamine(Wedeind等人，手稿正在准备中)。合成发夹结构的优点是各种非天然核苷酸碱基可以被结合到化学合成的RNA中，这本质上相当于蛋白质的定点突变。在本研究中，我们制备了包括G8I、G8U、G8(2，6二氨基嘌呤)在内的多种通用碱基突变体。在每一种情况下，突变的核酶在溶液中都是不活跃的，但可以通过添加外源镁(II)来挽救。我们认为Mg(II)发挥着重要的电子稳定作用，并建议在没有和存在(电子密度)Mn(II)的情况下收集数据集，以评估突变引起的构象变化并定位金属结合位点(S)。此外，还创造了影响剪刀位置上的环状折叠的突变体，包括：2`-脱氧(A-1)、2`-氨基(A-1)和2`-羟基(A-1)。这些突变体代表底物的不可切割类似物，在G8突变体的背景下，它们应该提供对2‘-亲核位置上氢键和糖折叠的“自然”模式的洞察。这项工作需要同步辐射，因为我们无法收集本源的高分辨率数据(大多数情况下超过3.0A)。高分辨率数据对于分配核糖环折叠、定位溶剂分子以及了解核酶立体化学如何影响其酶功能至关重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The hairpin ribozyme is a catalytic RNA that belongs to the family of small ribozymes. Members of this enzyme class perform a site-specific cleavage reaction that involves nucleophilic attack of a scissile phosphorus that leads to formation of a cyclic 2¿,3¿-phosphodiester and a free 5`-hydroxyl group. Among small ribozymes, the hairpin is unusual in that it does not require divalent metal ions for catalysis. Recent structural and chemical studies have suggested that the reaction proceeds via a general base/acid catalyst mechanism involving conserved bases G8 and A38. Residues contributing to electrostatic stabilization of the oxyphosphorane transition state are also required, but have not been identified fully. The goal of this proposal is to examine the structures of several mutant variants of hairpin ribozyme at the general base G8 position, as well as at the scissile bond (A-1). The applicant¿s lab has crystallized such mutants in the form of a 61-mer synthetic all-RNA construct that diffracted previously to 2.19 Angstroms resolution at beamline A-1 (CHESS). These results resulted in: (i) the highest resolution all-RNA structure of any naturally occurring ribozyme, (ii) identification of the inert 2¿-O-methyl group at the nucleophilic site, and (iii) locating the position of a stabilizing CoHexaamine in the S-turn structural motif (Wedekind et al, Manuscript in Preparation). The advantage of the synthetic hairpin construct is that a variety of non-natural nucleotide bases can be incorporated into the chemically synthesized RNA, which is essentially equivalent to site-directed mutagenesis of a protein. In this study, we have prepared a variety of general base mutants including G8I, G8U, G8(2,6 diaminopurine). In each case, the mutant ribozyme was inactive in solution, but could be rescued by addition of exogenous Mg(II). We believe the Mg(II) fulfills an important electronic stabilization role, and propose the collection of data sets in the absence and presence of (electron dense) Mn(II) in order to assess the conformational changes resulting from mutagenesis and to locate the metal binding site(s). In addition, mutants that effect the ring pucker at the scissile position have been created including: 2`-deoxy(A-1), 2`-amino(A-1), and 2`-hydroxyl(A-1). These mutants represent non-cleavable analogs of the substrate, which in the context of the G8 mutants should provide insight into the `natural` modes of hydrogen bonding and sugar puckering at the 2`-nucleophilic position. This work requires synchrotron radiation because we are incapable of collecting high-resolution data on the home source (beyond 3.0 A in most cases). High resolution data are critical to assign ribose ring puckers, to locate solvent molecules, and to understand how the ribozyme stereochemistry contributes to its enzymatic function.