Time Recovered Hydroxyl Radical Footprinting of RNA

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION (provided by applicant): In all organisms, RNA molecules are needed to translate genes into protein, and to turn specific genes on and off. The malfunction of controlling RNAs has been linked to diseases such as muscular dystrophy, Fragile X mental retardation, and certain tumors. In order to function normally, RNAs must fold into specific three-dimensional shapes and must assemble with particular proteins. For example, the ribosome contains two large RNAs and more than 50 proteins which must come together in precisely the right way. The goal of this research is to understand how RNAs fold up, and how they cooperate with proteins to form cellular "machines" such as the ribosome. The results will help understand how certain RNAs malfunction in human, genetic diseases, and will assist the design of antibacterial and antiviral drugs. We have previously used time-resolved hydroxyl radical footprinting, small angle scattering and biochemical methods to probe the folding pathway of ribozymes. Recently, we have used footprinting to follow the assembly of the SOS ribosome in real time. In the first aim, a stable bacterial group I ribozyme will be used as a model system to dissect the interactions that cooperatively stabilize RNA tertiary structure. The principles of RNA folding obtained from studies of ribozymes will be applied to assembly of SOS ribosomes in aims 2-4. The formation of RNA and RNA-protein interactions will be monitored by protection of the RNA backbone from hydroxyl radical cleavage and by changes in the fluorescence of labeled RNAs. Questions to be addressed are (1) how proteins recognize their binding sites and stabilize tertiary interactions in the rRNA, (2) whether proteins change the ribosomal RNA folding pathway, and (3) folding and assembly of the pre-168 rRNA. Long-term goals are to establish assays for ribosome assembly in situ, and to understand the link between the fidelity of RNA folding, assembly and processing of the pre-rRNA.