The ribosome is the key component in the final step of gene expression, protein synthesis. The focus of this research is the elucidation of the way in which ribosomal proteins contribute to the structure and function of two indispensable ribosomal domains: peptidyl transferase and GTP coupled translocation. The E. coli ribosome has been the objective of a variety of experimental approaches to define it overall structure, its peptidyl transferase center and its provision of binding sites for transient interactions with at least seven different protein factors, aminoacyl tRNA and messenger RNA. Comparative structural evidence points to a major role for RNA in the primitive translation machinery, but ribosomal proteins clearly play an indispensable role in the evolved ribosome. Protein crosslinking has provided a rough map that shows the relative locations of many of the 50S ribosomal proteins, in particular many of those in the two functional domains. Integration of this crosslinking map with three dimensional structure of the ribosome will be pursued by preparing monoclonal antibodies to proteins in functional domains and using them for immune electron microscopy. Antibodies against different epitopes within individual proteins will be made and used as probes of functional involvement in in vitro assays. A central strategy in this proposal is the use of in vitro mutagenesis of genes for ribosomal proteins to make structural alterations in key proteins. They are to be used in two ways: first, as an adjunct to the crosslinking approach used to the past by introducing cysteine residues at selected noncritical regions of a protein, so that the new SH group can be used as an identified site for crosslinking and the attachment of other structural probes for fluorescence and immune electron microscopy. This will be applied to the conserved, mobile, multi-copy protein of the translocation domain, L7/L12; second, to introduce random mutations into regions of proteins L2 and L16 of the peptidyl transferase domain, or to replace selectively certain histidine residues, in order to identify functionally important sites within these proteins. The first application involves the overproduction and purification of the mutant proteins, followed by reconstitution into the ribosome. Crosslinking to neighboring proteins will be accomplished either by disulfide bond formation or by the attachment to the SH group of a radioactive photoaffinity reagent that labels the neighboring protein with which it reacts. The second application involves screening strains expressing and altered protein for temperature sensitivity and analyzing the structural and functional properties of isolated ribosomes. In addition the overexpressed protein will be purified and reconstituted into ribosomes to determine its functional effect. The proposed research will greatly increase the resolution of the crosslinking map and lead to new insights into the mechanism by which proteins contribute to ribosome function.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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University of California Davis
Schools of Medicine
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