MAD EXPERIMENT OF GR DICER PROTEIN CRYSTAL; HIGH RESOLUTION DATA COLLECTION OF E

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们最近获得了DICER的晶体，这是RNA干扰(RNAi)途径中的一种关键酶，可以将X射线衍射到原子分辨率。这种大的二聚体蛋白质的分子结构将是理解RNA如何在体内被识别和切割成特定长度的产物的核心，这是通过靶向mRNA降解或翻译停滞进行RNA干扰的第一步。据我们所知，这是RNAi途径中第一种可获得高质量晶体的酶，其结构可能对我们理解RNAi的分子机制产生重大影响。含硒蛋氨酸的晶体目前已在手中，并准备用于MAD实验。真细菌信号识别粒子(SRP)由于其有限的复杂性，是研究其功能的高分辨率结构研究的极佳模型。我最近获得了功能性E.Coli SRP和SRP受体(SR)复合体的晶体，并收集了高达8A分辨率的初步衍射数据。一个高分辨率的结构模型，以及随后的生化实验，应该能够深入了解SRP的重要功能，包括信号肽是如何识别的，以及SRP和SR的GTPase活性是如何在不同的功能状态下调节的。

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. We have recently obtained crystals of Dicer, a key enzyme in the RNA interference (RNAi) pathway, that diffract X-rays to atomic resolution. The molecular structure of this large dimeric protein will be central to understanding how RNA is recognized and cleaved into specific length products in vivo as the first step in RNA interference by targeted mRNA degradation or translational arrest. To our knowledge this is the first enzyme in the RNAi pathway for which high-quality crystals are available, and the structure is likely to have a major impact on our understanding of the molecular mechanism of RNAi. Selenomethionine incorporated crystals are currently in hand and ready for use in MAD experiments. The eubacterial Signal Recognition Particle (SRP), because of its limited complexity, is an excellent model for high-resolution structural studies to address its function. I have recently obtained crystals of the functional E.Coli SRP and SRP receptor (SR) complex, and collected preliminary diffraction data up to 8 A resoltion. A high-resolution structure model, together with subsequent biochemical experiments, should provide insights into the important functions of SRP including how the signal peptide is recognized, and how the GTPase activities of SRP and SR are regulated at different functional states.