Protein-Dependent Splicing of Chloroplast Group II Introns

催化核糖核蛋白颗粒（RNP）是几个基本细胞过程的核心，包括蛋白质合成，tRNA加工和RNA剪接。RNA和蛋白质组分的贡献是不同的。在某些情况下，RNA亚基具有催化活性，并且蛋白质增强活性RNA结构的形成或稳定性。在其他情况下，RNA为催化活性蛋白质提供组装支架。 已知RNA和蛋白质分子在形成底物结合表面方面合作，并且原则上，它们可以合作形成活性位点。本项目利用具有“自我剪接”II组内含子的RNP来探索RNA与蛋白质之间的合作模式。II组内含子剪接的化学机制与剪接体中的化学机制相同，推测这两种剪接机制是从共同的祖先进化而来的。9个核编码的蛋白质，所需的剪接的各种子集的17组II内含子在玉米叶绿体中被确定。这些蛋白质为探索蛋白质和RNA在RNP组装和催化过程中如何合作提供了独特的工具。实验将集中在内含子RNP的子集包含一种称为CRS 2的蛋白质。CRS 2来源于细菌肽基-tRNA水解酶（PTH），并与两种密切相关的蛋白质CAF 1或CAF 2中的任一种形成异二聚体复合物。最近的数据表明，CRS 2/CAF复合物与内含子催化核心密切相互作用的方式，不同于以前研究的第二组内含子RNP的相互作用。结构和系统发育的数据表明，有趣的可能性，该地区的CRS 2来自PTH活性位点可能有助于剪接催化，这一概念是支持最近的生化研究内含子RNP架构，其中放置的CRS 2活性位点区域附近的分支点腺苷，启动剪接。实验将利用已建立的蛋白质表达系统和生物化学测定进一步剖析CRS 2/CAF/内含子RNP的结构，以测试CRS 2/CAF结合是否在催化核心组织RNA元件，并测试CRS 2是否直接有助于催化。所获得的结果可能会影响我们对剪接体，核糖体和其他催化RNP中RNA-蛋白质合作的理解。表达遗传信息的机制中最古老和最普遍的组成部分是RNA和蛋白质之间的大型复合物，这些大分子具有独特的生化和结构特征。在长期的共同进化过程中，RNA和蛋白质部分的贡献已经交织在一起，对这些分子之间可能的合作模式的理解是不完整的。该项目使用称为II组内含子RNP的催化RNA蛋白颗粒来探索这个问题。先前的结果表明，这些颗粒中的蛋白质发挥了独特的和更根本的作用，比以前记录的蛋白质在催化RNP。因此，所获得的结果可能会影响我们对其他大分子复合物中RNA-蛋白质合作的理解。高中生、本科生和研究生，包括少数民族/贫困学生，将通过参与该项目接受教育。

Catalytic ribonucleoprotein particles (RNPs) are at the core of several fundamental cellular processes, including protein synthesis, tRNA processing, and RNA splicing. The contributions of the RNA and protein components are varied. In some instances, RNA subunits harbor catalytic activity and the proteins enhance the formation or stability of the active RNA structure. In others, RNAs provide an assembly scaffold for catalytically-active proteins. RNA and protein molecules are known to cooperate in forming substrate binding surfaces and, in principle, they could cooperate to form an active site. This project uses RNPs harboring "self-splicing" group II introns to explore modes of cooperation between RNA and protein. The chemical mechanism of group II intron splicing is identical to that in the spliceosome and it is speculated that these two splicing machineries evolved from a common ancestor. Nine nucleus-encoded proteins that are required for the splicing of various subsets of the 17 group II introns in maize chloroplasts were identified previously. These proteins provide unique tools for exploring how proteins and RNAs cooperate during RNP assembly and catalysis. Experiments will focus on the subset of intron RNPs containing a protein called CRS2. CRS2 is derived from a bacterial peptidyl-tRNA hydrolase (PTH) and functions in heterodimeric complexes with either of two closely-related proteins, CAF1 or CAF2. Recent data suggest that CRS2/CAF complexes interact intimately with the intron catalytic core in a manner that differs from interactions in previously-studied group II intron RNPs. Structural and phylogenetic data suggest the intriguing possibility that the region of CRS2 derived from the PTH active site may contribute to splicing catalysis; this notion is bolstered by recent biochemical studies of intron RNP architecture, which place the CRS2 active site region near the branchpoint adenosine that initiates splicing. Experiments will take advantage of established protein expression systems and biochemical assays to further dissect the architecture of CRS2/CAF/intron RNPs, to test whether CRS2/CAF binding organizes RNA elements at the catalytic core, and to test whether CRS2 contributes directly to catalysis. The results obtained could impact our understanding of RNA-protein cooperation in the spliceosome, ribosome, and other catalytic RNPs. The most ancient and universal components of the machinery for expressing genetic information consist of large complexes between RNA and protein, macromolecules with distinct biochemical and structural features. The contributions of the RNA and protein moieties have become intertwined during a long period of co-evolution, and understanding of the repertoire of possible modes of cooperation between these molecules is incomplete. This project uses catalytic RNA-protein particles called group II intron RNPs to explore this issue. Prior results suggest that the proteins in these particles play a distinct and more fundamental role than has previously been documented for proteins in catalytic RNPs. Thus, the results obtained could impact our under-standing of RNA-protein cooperation in other macromolecular complexes underlying gene expression. High school, undergraduate and graduate students, including minority/underprivileged students, will be educated through their involvement in this project.