Post transcriptionally modified nucleotides in RNA are known to play a variety of functional roles, including stabilization of secondary and tertiary structure and enhancement of fidelity of intermolecular interactions in protein synthesis. By far, the greatest amount of information has been derived from studies of tRNA, although the details of these roles are in general not well understood. By contrast much less is known regarding structure-function relationships for post-transcriptional modifications in ribosomal RNAs e3ven though both large and small subunit rRNAs are now known to play direct roles in protein synthesis. It is proposed to apply new electrospray ionization LC/MS and LC/MS/MS based protocols to identify modified nucleotides and place them at precise sites in sequences of RNA from small and large ribosomal subunits, and of tRNAs. The proposal is focused on ribosomal RNAs and transfer RNAs of thermophilic bacteria and archaea, as models of understanding natural structural motifs for RNA stabilization. Modified oligonucleotides selected from the RNA modification maps determined will be synthesized for study of their biophysical properties compared with those of the corresponding unmodified sequences. These studies contribute to a clearer understanding of ribosome structure and function during protein synthesis, and of structural motifs that influence RNA stability. These issues are of potential importance in the design of antisense- and ribozyme-based therapeutic agents, in understanding the basis of resistance to ribosome-targeted antibiotics, and in expanding the limited knowledge based of natural modifications in RNAs involved in translation.
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