The structure of the ribosome and the mechanism of protein synthesis will be studied with techniques the determine the arrangement of the rRNA and its interaction with mRNA, tRNA, and ribosomal proteins. This study will lead to three dimensional models of the ribosome at higher resolution that is presently available. Site directed mutagenesis will be used as a complementary technique to investigate the function of the rRNA. These experiments will provide a better basis for understanding how mRNA is selected, how the rate and termination of protein synthesis are determined and how antibiotics disrupt ribosome function. The structural analysis will be done primarily with site specific psoralen (SSP),k a site-directed photoaffinity agents. Because we can control the placement of the distances that are measured will be approximately 5 A. UV cross-linking will also be used to monitor the overall configuration of the 16S rRNA. Both of these techniques will be very sensitive to structural changes.
The specific aims of this work are: 1. to determine the three dimensional structure of the 16S rRNA at the mRNA decoding site in active 30S subunits and in 70S ribosomes, 2. to determine whether subunit association, mRNA binding, and tRNA binding induce three dimensional structural changes in he 16S rRNA, 3. to determine how the wild type structure is altered by mutations in 16S rRNA that affect mRNA and tRNA binding. 4. to determine the path of the mRNA through the ribosome during translation.
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