THE HAIRPIN RIBOZYME: STRUCTURE, MUTAGENESIS AND MECHANISM OF ACTION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。发夹核酶是一种催化RNA，属于小核酶家族。这类酶的成员进行位点特异性切割反应，包括对可裂磷的亲核攻击，导致形成环状2¿，3¿-磷酸二酯和游离5 ' -羟基。在小的核酶中，发夹酶是不寻常的，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，反应是通过涉及保守碱G8和A38的一般碱/酸催化机制进行的。残基有助于氧磷过渡态的静电稳定也是必需的，但尚未完全确定。本提案的目的是研究发夹核酶的几个突变体在一般碱基G8位置的结构，以及在可剪键（A-1）。申请人的实验室已经将这种突变体结晶为61 mer合成的全rna结构，该结构先前在光束线a -1 （CHESS）衍射到2.19埃的分辨率。这些结果导致：(i)所有天然存在的核酶中分辨率最高的全rna结构，（ii）在亲核位点鉴定惰性的2¿- o -甲基，以及（iii）在S-turn结构基序中定位稳定的CoHexaamine的位置（Wedekind等人，Manuscript in Preparation）。合成发夹结构的优点是多种非天然核苷酸碱基可以被纳入化学合成的RNA中，这基本上相当于蛋白质的定点突变。在本研究中，我们制备了多种通用碱基突变体，包括G8I、G8U、G8（2,6二氨基嘌呤）。在每种情况下，突变核酶在溶液中都是无活性的，但可以通过添加外源Mg（II）来恢复。我们认为Mg（II）具有重要的电子稳定作用，并提出在没有和存在（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。此外，还产生了影响环状褶皱的突变体，包括：2 ' -脱氧（A-1）， 2 ' -氨基（A-1）和2 ' -羟基（A-1）。这些突变体代表了底物的不可切割类似物，在G8突变体的背景下，它应该提供对2 ' -亲核位置的氢键和糖皱缩的“自然”模式的见解。这项工作需要同步辐射，因为我们无法在主源上收集高分辨率数据（在大多数情况下超过3.0 A）。高分辨率的数据对于分配核糖环褶皱，定位溶剂分子以及了解核酶立体化学如何促进其酶功能至关重要。

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.