This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy (cryo-EM) and single-particle reconstruction. Protein biosynthesis is one of the most fundamental processes of life, but despite seminal work in solving the structure of the ribosome, this process is not well understood. Cryo-EM is uniquely suited to capture the molecule in functional states under close to native conditions. In this lab, several seminal discoveries regarding the dynamics of this process in both eubacterial and eukaryotic translation have been made in the past; among these the ratchet-like motion during mRNA-tRNA translocation and the large spring-like deformation of the aminoacyl-tRNA as it enters the ribosome during the aminoacyl-tRNA selection process. With the recent increase in resolution, structures of ribosomal complexes can be visualized with close to atomic resolutions. Special focus of this research program will be the structure and dynamics of the eukaryotic (parasites and mammalian) ribosomes.
The aims of the proposed studies are threefold: (1) to solve the structures, and characterize the functional dynamics, of ribosomes from four eukaryotic parasites known to cause debilitating human diseases; (2) to study the process of mammalian eukaryotic translation initiation by cryo-EM visualization of selected initiation complexes; (3) to study the process by which certain viruses (EMCV, polio) usurp the translational apparatus of the mammalian host to make protein required for assembly of new copies of the virus. Samples will be obtained from several collaborating labs that are specialized in eukaryotic translation. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of existing structural, kinetics, single-molecule FRET, and biochemical data. Density maps with resolutions equal or better than 3.0 A will be used for ab initio modeling of the atomic structure.
The research proposed using cryo-EM addresses structure and function of ribosomes in eukaryotic parasites causing Sleeping Sickness, Chagas Disease, Leishmaniasis, and Malaria, with the expectation that the specific information may lead to the development of more potent antibiotics. Another aim of the research addresses the fundamental mechanism of translation initiation in mammals, which is tightly regulated and whose dysfunction is implicated in a number of cancers. A third aim seeks an understanding of the mechanism by which certain viruses (e.g., encephalomyocarditis, polio) take over the human host's translation machinery.
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