Protein-RNA and protein-protein interaction in the immune defense system of prokaryotes

原核生物免疫防御系统中的蛋白质-RNA和蛋白质-蛋白质相互作用

• 批准号: 206973666
• 负责人: Professor Dr. Henning Urlaub
• 金额: --
• 依托单位: Institut für Klinische Chemie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/206973666?language=en
• 关键词: Protein; RNA; protein; interaction; immune

Mass spectrometry (MS) and its current applications address various questions of high relevance for cellular and structural biology. These applications include not only proteomic approaches, but also investigations of protein¿protein and protein¿ligand interactions, and protein structures. Here we will apply MS to various projects revolving around the immune defence system of prokaryotes, including protein-protein, protein-RNA, protein-DNA crosslinking of various CRISPR-Cas complexes, as well as to studies that monitor quantitative changes of prokaryotic proteomes upon phage invasion. The main goals of this project in a second funding period are to expand our MS-based investigations from the single protein-RNA complexes that we targeted during the first funding period to entire protein-protein, protein-RNA and protein-DNA complexes, which will enable a realistic structural modelling of these molecular machines. Mass spectrometry-based crosslinking approaches will supply us with sets of spatial constraints to allow for molecular modelling of the complexes, especially where structural information acquired by e.g. X ray crystallography is not, or only partially available. Furthermore, the methods that we have developed during the first funding period will allow us to apply our protein-RNA crosslinking approach to in vivo samples, i.e. to entire prokaryotic cells in order to identify protein-RNA contact sites in living cells. By relating the crosslinking results to quantitative proteome studies of prokaryotes under phage invasion, we aim to develop a detailed understanding of how protein-RNA(DNA) interactions in the CRISPR-Cas system contribute to the integration of the phage genome into the host genome.

质谱（MS）及其当前的应用解决了与细胞和结构生物学高度相关的各种问题。这些应用不仅包括蛋白质组学方法，还包括蛋白质、蛋白质和蛋白质配体相互作用以及蛋白质结构的研究。在这里，我们将MS应用于围绕原核生物免疫防御系统的各种项目，包括各种CRISPR-Cas复合物的蛋白质-蛋白质，蛋白质-RNA，蛋白质-DNA交联，以及监测噬菌体入侵后原核蛋白质组定量变化的研究。该项目在第二个资助期的主要目标是将我们基于MS的研究从我们在第一个资助期针对的单一蛋白质-RNA复合物扩展到整个蛋白质-蛋白质，蛋白质-RNA和蛋白质-DNA复合物，这将使这些分子机器的现实结构建模成为可能。基于质谱的交联方法将为我们提供一组空间约束，以允许复合物的分子建模，特别是在通过例如X射线晶体学获取的结构信息不可用或仅部分可用的情况下。此外，我们在第一个资助期内开发的方法将使我们能够将我们的蛋白质-RNA交联方法应用于体内样品，即整个原核细胞，以鉴定活细胞中的蛋白质-RNA接触位点。通过将交联结果与噬菌体入侵下原核生物的定量蛋白质组研究相关联，我们的目标是详细了解CRISPR-Cas系统中的蛋白质-RNA（DNA）相互作用如何有助于噬菌体基因组整合到宿主基因组中。