Triman 9726951 A detailed understanding of the roles of specific nucleotides in the structure, assembly and processing of the bacterium Escherichia coli's 16SrRNA and 23S rRNA ribosomal RNAs is the ultimate goal of these studies. The general approach outlined here involves the study of seven specific base pairs in 165 rRNA and one specific base pair in 23S rRNA. These base pairs represent sites in which conditional mutations have been identified that confer loss of expression of a selectable marker (spectinomycin-resistance or erythromycin-resistance) Each of the mutations disrupt a G-C base pair in or near a region of rRNA involved in interaction with a ribosomal protein. Genetic studies of these mutations will involve (1) introduction of compensatory second-site alterations by site-directed mutagenesis, (2) determination of the phenotype conferred by A-U replacement of each specific G-C base pair disrupted by the primary mutation, and (3) determination of the phenotype conferred by the compensatory alteration in the absence of the primary mutation. Restoration of base pairing at a given position is predicted to confer full drug resistance if the mutant phenotype is due to disruption of pairing. Failure to restore drug resistance is taken as evidence that the primary mutation has disrupted some other structural interaction in which the site is involved. Thus, the genetic approach can be used to either confirm the importance of specific base paired nucleotides in the secondary structure of rRNA or to identify unanticipated structural roles for those nucleotides. These genetic studies are designed to determine the importance of specific nucleotides by assessment of the structural and functional impairments of rRNA molecules resulting from expression of base substitutions at these positions. Databases of ribosomal RNA mutations identified to date are also maintained. On-line access to these database is being improved by implementing a query form and a data submission form and by provid ing a Webserver. In addition, undergraduate students will be involved in all aspects of this project. This research and database is important because the ribosome is responsible for the translation of messenger RNA, the initial product of genes, into proteins in all living organisms.
