本文研究对象是多亚基蛋白质复合物，以复合物分子的三维结构解析与功能探讨为目的，以X射线晶体学作为研究手段。该蛋白质家族成员在细胞中参与了多个重要的RNA代谢通路，在原核生物、古细菌、真核生物中很保守。在真核细胞中，该家族蛋白质的不同成员可以识别不同的RNA序列，形成核酸蛋白质复合物，执行不同的生物功能。为了获得可结晶的高浓度高纯度的蛋白质复合体，以大肠杆菌作为表达系统，通过体外重组表达和纯化得到了排列组装正确的蛋白质复合物。通过蛋白质结晶并在X射线衍射仪和同步辐射衍射仪收集数据之后，用多个软件对衍射图谱进行分析，解析了两个重要的蛋白质复合体的分子结构。该分子结构中不同亚基的排列顺序与先前文献根据生化实验和细胞实验得出的排列顺序一致。本文还通过合成以及体外转录和非变性纯化的方法获得蛋白质复合体所识别的特异RNA序列。再通过多种手段证明RNA与蛋白质能形成稳定的复合物。这也证明了得到的蛋白质复合物状态是具有功能的。本课题解析的两个蛋白质复合物中，多数的蛋白质亚基相同。只有一个亚基的不同，导致了两个蛋白质复合物的细胞定位、识别的RNA序列以及参与的细胞通路完全不同。通过结构的解析，可以从分子水平解释两种蛋白质复合物功能的差异。为该蛋白质超家族的进化研究提供了重要信息。多年来，关于该蛋白质复合体的研究工作已经报道了很多。但是获得复合体晶体结构的尝试都失败。这篇论文提供了新的蛋白质复合物共表达与纯化的手段，不仅为此领域提供了解决问题的方案，还为其它蛋白质复合物的结构生物学研究提供了新的研究手段。在解析了两个蛋白质复合物分子结构的基础上，对于其识别的RNA序列和其它蛋白质分子进行了探索性实验。体外重构了大的核酸蛋白质复合物，为该领域中进一步的结构生物学研究提供了一定的基础。
The thesis focused on protein complexes involved in several RNA metabolism pathways, which are conserved from prokaryotic cells to archea and eukaryotic cells. To illustrate the 3-dimensional structures of protein complexes and the molecular mechanism on how the protein complexes recognize specific RNA sequence, crystallography approach is applied. The target proteins can bind to RNA and form RNA-protein complex and play important roles in the cell.To obtain the protein complex with high concentration and high purity in correct order, protein co-expression is E.coli. Protein crystals grew up in certain conditions. With diffraction patterns of the proteins on X-ray machine and synchrotron radiation, two 3D structures of the protein complexes are solved.The two structures of protein complexes share most of the subunits, with only one subunit different. But the two complexes are located differently, recognize different RNA sequences and have completely different functions.The specific RNA is purified in non-denatured way from in vitro transcription system. With several different approaches, it is verified that the protein complex can form stable complex together with the RNA. Another protein component, which has interaction with the target protein complex and the specific RNA sequence, is expressed and purified. In vitro reconstitution of the larger complex is carried out, hopefully leading to another important structure in the pathway.This thesis provides a new method for protein complexes expression and purification, as well as their crystal structures. With these information, the explorations on related RNA metabolism pathway can be carried out. Important breakthroughs on structural and functional study can be expected in near future.
