STRUCTURAL STUDIES OF THE 50S RIBOSOMAL SUBUNIT WITH SUBSTRATE ANALOGUES

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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. The 50S ribosomal subunit from Halocarcula marismortuii has provided atomic level resolution of structure-function relationships in protein synthesis. In recent years, we have solved the structures of the large ribosomal subunit complexed with several small analogues that represent different steps along the peptidyl transferase reaction pathway, including each individual substrate alone, a putative transition state intermediate, and the products of the reaction. Last year, we collected preliminary data on crystals of 50S complexed with analogues representing the two substrates of the reaction bound simultaneously (in A and P-sites). Experiments have been performed with RNA substrates and substrates that have been chemically modified to aid in determining the roles of particular atoms in peptidyl transferase. We shall obtain new data to solve the high resolution structure of the 50S ribosomal subunit with substrates bound in the A- and P- site, to determine the exact conformation of the ribosomal active site directly before peptidyl transfer. In addition, we have obtained substrates with substitutions at positions critical to the peptidyl transferase reaction. Solving the structures of these modified substrates bound to the ribosome will enable us to learn the roles of these groups in peptide bond formation, and help determine the source of the ribosome's catalytic power. In addition to pursuing the all-RNA version of the previously solved transition state analogue, we will also be using chiral intermediate analogues to study the reaction. Determining the structure of a chiral transition state analogue will indicate the stereochemistry of the nucleophillic attack. Lastly, we will determine the structure of the 50S with the largest possible fragment of deacylated tRNA bound to its E-site. This structure may allow us to understand the mechanism by which tRNAs are passed from the P-site to the E-site during the translocation cycle.