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).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029274-04A1
Application #
3276837
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1981-01-01
Project End
1988-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Ohio State University
Department
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
Kim, J H; Marshall, A G (1990) Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements. Biochemistry 29:632-40
Kim, J H; Marshall, A G (1990) Dynamic structure of bacterial ribosomal 5S RNA helices II and III of B. megaterium 5S RNA. Biochem Biophys Res Commun 169:1068-74
Wu, J J; Marshall, A G (1990) Wheat germ 5S ribosomal RNA common arm fragment conformations observed by 1H and 31P nuclear magnetic resonance spectroscopy. Biochemistry 29:1730-6
Wu, J J; Marshall, A G (1990) 500-MHz proton NMR evidence for two solution structures of the common arm base-paired segment of wheat germ 5S ribosomal RNA. Biochemistry 29:1722-30
Lee, K M; Marshall, A G (1987) Morpholino spin-labeling for base-pair sequencing of a 3'-terminal RNA stem by proton homonuclear Overhauser enhancements: yeast ribosomal 5S RNA. Biochemistry 26:5534-40
Li, S J; Wu, J J; Marshall, A G (1987) 500-MHz proton homonuclear Overhauser evidence for additional base pair in the common arm of eukaryotic ribosomal 5S RNA: wheat germ. Biochemistry 26:1578-85
Lee, K M; Marshall, A G (1986) High-speed preparative-scale separation and purification of ribosomal 5S and 5.8S RNA's via Sephacryl S-300 gel filtration chromatography. Prep Biochem 16:247-58
Lee, K M; Marshall, A G (1986) Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements. Biochemistry 25:8245-52
Li, S J; Marshall, A G (1986) Identification and assignment of base pairs in the ""tuned helix"" of intact and ribonuclease T1 cleavage fragments of wheat germ ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements. Biochemistry 25:3673-82
Chen, S M; Marshall, A G (1986) Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements. Biochemistry 25:5117-25

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