Probing the RNA structure within the yeast 5 S RNA.L1a protein complex by fluorescence and enzymatic digestion.
Conformational states of the ribosomal 5 S RNA molecule and its associated protein L1a in the yeast RNA-protein (RNP) complex were determined using controlled RNase T1 digestion in conjunction with fluorescence probes, ethidium bromide and bisanilinonaphthalenesulfonic acid. Fluorescence measurements indicated that the RNA molecule in the RNP complex appeared to exhibit a slightly lower degree of secondary structure than that in the free form. Controlled digestion of the intact RNP complex with RNase T1 resulted in an initial increase in ethidium fluorescence followed by a gradual decrease. In free RNA, a similar profile, except that a larger increase in ethidium fluorescence at the initial stage of digestion, was observed. During digestion of the RNP complex, increases in bisanilinonaphthalenesulfonic acid fluorescence and in light scattering were observed. These findings implied that as regions of the 5 S RNA molecule were perturbed, hydrophobic regions in the protein became exposed. Polyacrylamide gel analysis of the digestion products revealed a temporal appearance of discrete RNA fragments. Sequence analysis of these fragments generated information about the structural arrangement of the RNA molecule within the RNP complex. Results from the present investigation indicate that interactions between the 5 S RNA and protein L1a can stabilize functionally relevant conformations of the components that are individually labile. Properties of the separated components also suggest that special conditions, such as those suggested by Steitz et al. (Steitz, J. A., Berg, C., Hendrick, J. P., LaBranche-Chabot, H., Metspalu, A., Rinke, J., and Yario, T. (1988) J. Cell Biol. 106, 545-556) may be involved for these components to associate during ribosomal assembly.
D001483:Base Sequence; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008027:Light; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012335:RNA, Ribosomal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D006163:Ribonuclease T1; D012269:Ribosomal Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D005656:Fungal Proteins; D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins; D006163:Ribonuclease T1