RIBOSOMAL FRAMESHIFTING AS A PROBE OF 5S RRNA FUNCTION

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DESCRIPTION (Applicant's abstract): The ribosome is the central component of an extremely accurate cellular protein synthetic apparatus. Its job is to rapidly and accurately decode mRNAs by reading three base "codons." Although it is exquisitely designed to maintain translational reading frame, there are several naturally occurring instances in which this process has been subverted, one of which is called programmed ribosomal frameshifting (PRF). Such exceptions to the rule provide a powerful set of tools to elucidate the molecular mechanisms underlying the normal maintenance of translational reading frame. With the advent of molecular genetics, it has been possible to create and examine the effects of mutants of individual ribosomal components on different ribosome-associated functions using specialized assay systems. The application of these new tools to classic biochemical methods are leading to a deeper understanding of the roles that many of the ribosomal proteins and ribosomal RNAs (rRNAs) play in determining how ribosomes maintain translational reading frame. It is now clear that the rRNAs are the central players in the reactions catalyzed by ribosomes, and that the individual rRNAs are actively involved in different ribosome functions. However, although it is highly conserved throughout evolution, the precise function of the ubiquitous 5S rRNA remains undetermined. Our demonstration that mutants of the yeast 5S rRNA (called mof9) can alter PRF efficiencies provides us with a unique platform from which investigations into the function of 5S rRNA can be launched. Preliminary results are consistent with recent structural reports suggesting a role for 5S rRNA peptidyltransferase center and A-site associated functions. The studies proposed herein will use PRF as a primary indicator of function to genetically and biochemically characterize 5S rRNA. Two complementary yeast systems will be utilized to monitor the affects of specific 5S rRNA mutants on PRF efficiencies, nonsense suppression, and cell growth and viability. Biochemical studies will be performed to test for specific functional and structural defects due to the mof9 mutants. Moreover, given the demonstrated physical interactions between 5S rRNA and ribosomal protein L5 (L5), we will follow up on preliminary studies to determine how this protein also contributes to translational reading frame maintenance. The proposed research will serve to further our understanding of this ubiquitous yet little understood rRNA and will place us in the unique position to link functional aspects of 5S rRNA to its structure within the ribosome.