Studies of RNA-protein interactions in the yeast 5 S ribonucleoprotein particles by fluorescence and tritium exchange. Implications for ribosomal assembly.
Studies of the conformational properties of the yeast 5 S RNA-protein complex were initiated in an attempt to understand loss of ability of its individual protein and RNA components to reassociate. The 5 S RNA-L1a protein complex from 60 S ribosomal subunits of Saccharomyces cerevisiae could be dissociated by high concentrations of magnesium. The degree of dissociation could be monitored by polyacrylamide gel electrophoresis. The complex was completely dissociated at about 390 mM magnesium, but was stable at 4 degrees C in 25 mM EDTA up to 48 h. The overall conformation of the complex was monitored using tritium exchange. The tritium exchange behavior was dramatically changed as the complex was dissociated. To determine contribution of each component to the observed overall change reflected in the tritium exchange behavior, ethidium bromide (EtBr) and bis-anilinonaphthalene-sulfonic acid fluorescence were used to monitor the RNA and the protein moiety, respectively. Upon dissociation of the complex, the fluorescence intensity resulting from EtBr binding to RNA decreased, whereas the intensity due to bis-anilinonaphthalene-sulfonic acid binding to the protein increased. Turbidity was observed during dissociation of the complex. These results indicate that disruption of interactions between the 5 S RNA and protein L1a resulted in an exposure of solvent-accessible apolar regions in the protein molecule. Such exposure led to insolubility of protein and irreversibility in interaction between individual components. Properties of the separated components also suggest that special conditions may be required for these components to associate during ribosomal assembly.
D008970:Molecular Weight; D012335:RNA, Ribosomal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D012261:Ribonucleoproteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D014316:Tritium
D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D012261:Ribonucleoproteins; D014316:Tritium