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

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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.