Our main long-range goal continues to be a detailed understanding of the structure and function of ribosomes. We are studying the closely related areas of ribosome assembly, RNA-protein interaction, RNA structure and ribosome evolution. A central theme of this research, which we set forth in the early 1970's, is that ribosomal RNA is itself involved in ribosome function, and not simply a scaffold for the assembly of ribosomal proteins. Our experimental approaches are mainly biochemical, although certain aspects of this proposal also invoke biophysical and genetic methods. We also make extensive use of computer methods, for example in comparative sequence analysis and in computer modeling of higher order rRNA structure. Demonstration of ribosomal function using naked rRNA would be a definitive demonstration of the correctness of the rRNA paradigm. One project is aimed at showing that naked 23S rRNA can catalyze peptide bond formation, using the """"""""fragment reaction"""""""". Another approach to this general question is to show that tRNA, antibiotics or protein synthesis factors interact with naked rRNA in the same way that they interact with rRNA in ribosomes. Following our previous studies on the identification of functional sites in rRNA we plan to complete the survey of ribosomal functions, using our chemical probing/primer extension method. These studies will investigate interactions between rRNA and specific mRNAs, initiation factors and termination factors. The connection between subunit association, tRNA binding and translocation will be tested by two-dimensional Serwer gels, and by low-angle X-ray scattering. Details of tRNA-rRNA interaction will be studied using tRNAs with specifically altered structures, and by damage/selection experiments. The three-dimensional model for 16S rRNA will be updated and refined, and modeling of 23S rRNA will be initiated. Studies on smaller ribosomal sub-structures will be done, by transcription of rRNA gene fragments using T7 RNA polymerase. Finally, new methodologies will be pursued, as always. New base-specific and backbone-specific RNA probes will be developed for use with our primer extension method. Another promising approach will be to attach free radical-generating groups to functional ligands. The radicals will attack the neighboring rRNA within a sphere of a few angstroms, allowing us to identify rRNA structural and functional neighborhoods. This will also provide a new and critical test of our proposed three-dimensional model for 16S rRNA.
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