Isolation, characterization, and stability of the 30S ribosomal RNA complex from HeLa cells

• Published in: Biochemistry (Easton) Vol. 14; no. 2; pp. 338 - 346
• Main Authors: Ray, Durwood B, Franzen, James S, Vaughan, Maurice H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.01.1975
• Subjects: Centrifugation, Density Gradient; Chromatography, Gel; Drug Stability; Electrophoresis, Polyacrylamide Gel; HeLa Cells - analysis; Kinetics; Osmolar Concentration; RNA, Neoplasm - isolation & purification; RNA, Ribosomal - isolation & purification; Temperature
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00673a021

The HeLa 30S rRNA molecule (historically designated 28S rRNA) can be dissociated into two components, a 7S rRNA and a large rRNA component which we call 29S rRNA. To evaluate conformational differences between the 30S rRNA complex and the isolated 29S rRNA component of the complex, viscosity, sedimentation velocity, circular dichroism, and ultraviolet absorption measurements with the two species were performed. Sedimentation equilibrium studies were also carried out with the 30S rRNA complex. In addition, the kinetics of the reaction which dissociates the 30S rRNA complex were characterized. The removal of glycogen-like molecules by cetyltrimethylammonium bromide prescipitation of the rRNA and the preequilibration of rRNA with solvent by Sephadex column chromatography were found to be essential for reproducibility. The s20,2o values for the 30S rRNA complex and the isolated 29S rRNA were determined from the experimental data obtained at various rRNA concentrations as 29.89 plus or minus 0.40 and 29.09 plus or minus 0.14, respectively. The corresponding intrinsic viscosity values were 74 plus or minus 5 and 67 plus or minus 5 cm3/g, respectively. The optical properties of the 30S rRNA and 29S rRNA were not significantly different. These results indicate that there is no significant conformational difference between 30S rRNA and 29S rRNA under the conditions studied. We conclude from the sedimentation equilibrium data that the molecular weight of 30S rRNA is 2.1 x 10-6. From the kinetic data, the 30S rRNA dissociation appears to be an irreversible, cooperative, and ionic strength dependent reaction which at an ionic strength of 0.051 has an activation enthalpy of 123.5 kcal/mol and an activation entropy of 0.21 kcal/(mol deg).