Biochemical analysis of a 5S rRNA-associated sub-particle from trypsinized eukaryotic ribosomes.
On limited trypsinization, eukaryotic ribosomes released sub-particles that comprised a 5S rRNA molecule and two peptides (a 32 kDa and a 14 kDa). By tryptic finger-printing and amino-terminal sequence analysis, these two peptides were determined to be derived from large subunit ribosomal protein L5 (rpL5). The 32 kDa peptide represents the rpL5 protein minus the amino terminal eight residues and the carboxyl terminal ends (approximately 21 residues), whereas the 14 kDa peptide comprised near the amino-terminal region. The time course of ribosome trypsinization revealed that the two peptides were released kinetically. The indicated that the amino and carboxyl terminal ends of rpL5 were the first to be hydrolyzed, suggesting that the two ends of the rpL5 protein were exposed on the surface of ribosomes. Exposure of the carboxyl-terminal end was confirmed by use of an anti-L5c antibody raised against the carboxyl terminal region of rpL5. The kinetic data also revealed that the nearby amino terminal region of rpL5 (represented by the 14 kDa peptide) was the last part of rpL5 to be hydrolyzed, which was considered to be the 5S rRNA binding site.
Lin
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Institute of Genetics, National Yang-Ming University, Taipei, Taiwan.
Liu
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Lin
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D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008099:Liver / Q000737:chemistry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D012341:RNA, Ribosomal, 5S / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin
D010446:Peptide Fragments; D012341:RNA, Ribosomal, 5S; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; C029742:ribosomal protein L5; D014357:Trypsin