Probing the yeast 5 S RNA-protein complex by fluorescence and controlled proteolytic digestion.
The nature of the interaction between the RNA and the protein component in the yeast 5 S rRNA-L1a complex was assessed using fluorescence and controlled proteolytic and RNase digestion. (a) Influence of L1a on the RNA conformation was monitored by ethidium fluorescence and controlled RNase T1 digestion. The complex was digested with alpha-chymotrypsin, Staphylococcus aureus protease V8, subtilisin, or trypsin. Both termini of L1a in the complex were readily accessible to proteases. Proteolytic digestion of the complex resulted in a reduction in fluorescence intensity if ethidium was added after proteolysis. No change was observed when ethidium was allowed to react with the complex prior to proteolysis. Neither the rate of proteolysis nor the resultant peptide pattern was affected by the presence of ethidium. T1 digestion of intact RNP and trypsin-treated RNP produced different oligonucleotide patterns. Both the fluorescence and the T1 digestion data suggest that the conformation of the RNA moiety was influenced by the protein. (b) Influence of the RNA molecule on L1a conformation in the complex was monitored by limited proteolysis. Whereas the protein in the complex was relatively sensitive to proteases, free protein was completely resistant to digestion under identical conditions. The trypsin sensitivity of L1a in complexes containing different truncated 5 S RNA molecules was studied also. Upon removal of residues 31-49 of the 5 S RNA molecule, L1a in the complex became resistant to proteolysis. These results are interpreted in a model in which specific regions of both the RNA and the protein are involved in the interaction.
Yeh
L C
LC
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Lee
J C
JC
D002918:Chymotrypsin / Q000378:metabolism; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010449:Peptide Mapping; D012335:RNA, Ribosomal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
D005656:Fungal Proteins; D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D016601:RNA-Binding Proteins; C489268:RPL5 protein, S cerevisiae; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D002918:Chymotrypsin; D014357:Trypsin