Structure of a ribozyme catalytic center

描述（由申请人提供）：对于真核细胞中的大多数基因，初级转录物的剪接是由一个兆道尔顿分子机器剪接体进行的，剪接体由100多种蛋白质和5个小核rna （snrna）组成。然而，这种剪接反应也是由II组内含子RNA酶（核酶）进行的，而不需要蛋白质。这些核酶与剪接体有着惊人的结构和机制相似性，这使它们成为研究剪接基本生物化学的一个有吸引力的模型。现在已经确定结构域1到3 (D123)，结构域5 （D5）和外显子底物形成了II族内含子催化所必需的最小活性位点。唯一已知的活性位点组分的结构是孤立的D5。然而，D5不能单独模拟活性催化结构。因此，在D123和底物的背景下研究D5的结构对于提高我们对催化的原子基础的认识至关重要。为此，我们将幽门杆菌的II族内含子重构为一个新的三方核酶系统，该系统与以前的模型系统有一些重要的区别，这使其成为结构研究的一个有吸引力的目标。我们假设D5和底物的内部凸起形成了催化位点的关键部分。我们将应用强大的生物物理和生化工具来获得详细的结构洞察到这个核酶的最小催化核心。我们的总体具体目标是：1。确定D5凸起缺失突变体d5a25delta催化缺陷的分子基础。2. 利用核磁共振、诱变和紫外交联绘制D5凸起突变体和D123在存在和不存在底物的情况下的结合界面3。为了获得最小活性位点的原子视图并确定对结合和催化重要的特征，使用NMR和4确定D5和D5凸起突变体与D123（和或D123片段）和底物结合的结构。用生化分析验证结构。意义：我们预计这些研究的完成将为解释II族内含子的广泛生化和遗传数据提供一个原子框架，以扩展我们对催化的理解，这些可能为细胞剪接中RNA相互作用的细节提供第一个证据，并为开发II族内含子作为潜在的核酶治疗方法提供新方法。

DESCRIPTION (provided by applicant): For most genes in eukaryotic cells, splicing of the primary transcript is undertaken by a megadalton molecular machine, the spliceosome, consisting of over 100 proteins and five small nuclear RNAs (snRNAs). However, this splicing reaction is also carried out by the Group II intron RNA enzymes (ribozymes) without the need of proteins. Such ribozymes share striking structural and mechanistic similarities to the spliceosome, which makes them an attractive model for studying the basic biochemistry of splicing. It is now well established that domains 1 through 3 (D123), domain 5 (D5) and exon substrates form the minimal active site essential for catalysis by group II introns. The only known structure of the active site component is D5 in isolation. Yet D5 alone cannot mimic the active catalytic configuration. Examining the structure of D5 in the context of D123 and substrates is therefore critical for advancing our knowledge of atomic basis for catalysis. To this end we have reconstructed a group II intron from Pylaiella littoralis into a novel tripartite ribozyme system that has some important difference from previous model systems and that makes it an attractive target for structural studies. We hypothesize that the internal bulge of D5 and substrate form a key part of the catalytic site. We will apply powerful biophysical and biochemical tools to obtain detailed structural insight into the minimal catalytic core of this ribozyme. Our overall specific aims are: 1. Determine the molecular basis for the catalytic deficiency of a D5 bulge deletion mutant-D5A25delta. 2. Map the binding interface between D5 bulge mutant and D123 in the presence and absence of substrates using NMR, mutagenesis, and UV crosslinking 3. To obtain an atomic view of the minimal active site and to identify the features that are important for binding and catalysis, determine the structure of D5 and D5 bulge mutant bound to D123 (and or fragments of D123) and substrate using NMR, and 4. Validate the structure using biochemical assays. Significance: We anticipate that completion of these studies will provide an atomic framework for interpreting the extensive biochemical and genetic data on group II introns to extend our understanding of catalysis, these might provide the first evidence for the details of RNA interactions in cell splicing, and suggest novel approaches to developing group II introns as potential ribozyme therapeutics.