Interaction of 5SrRNA-L5 protein complex, methionyl-tRNA, and methionyl-tRNA synthetase in the macromolecular ARS complex.
Rat liver cytosol was incubated with a trace amount of rat liver 5SrRNA which was highly labeled at the 3'-end with cytidine 3',5'-[5'-32P]biphosphate, and with [35S]methionine in the presence of ATP mixture, and then with an antibody against ribosomal protein L5. The mixture was analyzed by protein A-Sepharose chromatography. The following results were obtained. (i) The eluate with glycine-HCl buffer (pH 3.0) from the protein A-Sepharose column contained an overlapping peak of 32P- and 35S-radioactivities. In a control experiment using the same amount of 32P-labeled Escherichia coli 5SrRNA with the same specific activity, no fraction of the eluate contained 32P-radioactivity. (ii) The fractions containing both 32P- and 35S-radioactivities from the protein A-Sepharose column were crosslinked by UV irradiation. The products was subjected to PAGE, and RNA in each gel slice was eluted and purified. The fraction containing both 32P- and 35S-radioactivities was present in a region of somewhat higher molecular weight than that of 5SRNP, whereas very low 32P- and 35S-radioactivities were present in this region in the control experiment without UV irradiation. This finding suggested that [35S]methionyl-tRNA interacted with 32P-labeled 5SRNP. (iii) The fraction containing overlapping 32P- and 35S-radioactivities described above was subjected to Sephadex G-150 chromatography. The component containing both radioactivities was distributed in the region corresponding to molecular weights of 10,000 to 250,000 with a peak at about 200,000, suggesting the presence of a complex containing Met-RS (Mr 108,000), 5SRNP (Mr 74,000), and methionyl-tRNA (Mr 25,000). Furthermore, this fraction showed definite Met-RS activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Ogata
K
K
Institute for Gene Expression, Dobashi Kyoritsu Hospital, Ehime.
Kurahashi
A
A
Ohno
R
R
Takahashi
K
K
Terao
K
K
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D002845:Chromatography / Q000379:methods; D003432:Cross-Linking Reagents / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D003911:Dextrans; D004926:Escherichia coli / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D012329:RNA, Bacterial / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D051381:Rats; D012269:Ribosomal Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000906:Antibodies; D003432:Cross-Linking Reagents; D003911:Dextrans; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D012329:RNA, Bacterial; D012341:RNA, Ribosomal, 5S; D012358:RNA, Transfer, Met; D012269:Ribosomal Proteins; C029742:ribosomal protein L5; C025614:sephadex; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase