The objectives of this work are to: 1) determine the three dimensional structure of the 16S rRNA at the mRNA decoding site and in the tRNA binding sites in active 30S subunits. 2) determine whether tRNA binding induces three dimensional structural changes in the 16S rRNA. 3) determine how the native structure is altered by mutations in 16S rRNA that produce 30S subunits that are not able to bind tRNA as well as the wild type sequence. 4) determine the path of the mRNA through the ribosome during translation. The structural analysis will be done primarily with a site directed photoaffinity agent placed at a series of specific sites in the 16S rRNA. Psoralen and UV crosslinking will also be used to monitor the overall configuration of the 16S rRNA. These experiments will provide new information about the three dimensional configuration of the RNA segments close to the decoding site at greater resolution than is presently available and will also provide a sensitive method to detect structural changes. It is expected that this information will help clarify the mode of the action of the 30S subunit during tRNA binding and protein synthesis.
