"This project will elucidate the structure and mechanism of ribosome function through the determination of high resolution crystal structures of human ribosome functional complexes. Currently, the only high resolution crystal structures available for the ribosome functional complexes are bacterial ribosomes containing mRNA, tRNAs in E, P and A sites and several translation factors. Recently, we have determined at medium resolution the first eukaryotic ribosome structure. Saccharomyces cerevisaiae ribosomes have been chosen for the first step of the study because they have been extensively investigated in our laboratory by biochemical methods and by x-ray analysis.
Step One: We will improve the quality of the crystals and determine the ribosome structure of yeast at atomic resolution (about 3Å resolution). These conditions will be used for an investigation of the ribosome functional complexes in order to better understand the detailed mechanism of the translocation of large molecules (tRNA and mRNA) on the ribosome.
Step Two: Ribosomes from human HeLa cells will be investigated via crystallization and x-ray structure determination. Ribosome functional complexes developed on yeast systems will be used as models for the creation and structural investigation of human ribosome complexes. Available biochemical data will provide a framework for the interpretation of structural information which will be obtained.
The aims:
To determine the atomic resolution crystal structure of the yeast ribosome as a model for all eukaryotic ribosome x-ray studies.
To obtain crystals and determine the structure of 80S ribosome from HeLa cells.
To determine the structure of the ribosome complexes with mRNA and tRNA in ratcheted and non-ratcheted states in order to describe the mechanism of translocation.
To determine structure of the ribosome initiation complex with an internal ribosomal entry site mRNA.
To obtain crystals and to determine the structures of 40S initiation complexes."