Objectives: A concerted series of site-selective physical measurements on ribosomal RNA's grown and isolated from both eukaryotic and prokaryotic organisms is designed to identify specific secondary and tertiary architectural features of 5S RNA and 5.8S RNA in solution. The results should make it possible to establish a secondary base-pairing pattern general to all known 5S RNA and 5.8S RNA primary nucleotide sequences. This universal secondary structure could then form a basis for tracing phylogenetic pathways over an extremely long evolutionary time scale, since the presence and function of 5S RNA is so highly conserved for all organisms from which it has been isolated. Methods: Optical techniques (ultraviolet, Fourier transform infrared and Raman spectroscopy) give the approximate total number of base pairs and the relative and absolute numbers of GC and AU pairs. Proton NMR at 500 MHz gives the specific number, types and sequence (e.g., AU followed by GC) of base pairs (including GU pairs) from intra-and inter-pair homonuclear Overhauser enhancements. Stepwise unfolding of the molecule with temperature is monitored by 1H and 31p NMR, FT-IR, ESR and differential scanning microcalorimetry. ESR spin labeling at the 5S RNA terminus, and at specific residues aids in assignment of NMR signals and in determination of local flexibility at those sites. Nucleotide bases exposed at the RNA surface are detected by effects of paramagnetic agents or chemically induced dynamic nuclear polarization (CIDNP) effects on 1H NMR signals. Efforts to crystaliize several 5S RNA's (for eventual X-ray diffraction studies) are in progress. Thermodynamic stabilities will be computed theoretically for all 80-odd primary sequences, for each of several proposed base-pairing schemes. Finally, all methods are applied to 5S RNA from several species (e.g., B. subtilis, E. coli, Neurospora crassa, wheat germ, and yeasts (Saccharomyces carlsbergensis; Torulopsis utilis).
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