INTERACTION OF 5SRRNA-L5 PROTEIN COMPLEX, METHIONYL-TRANSFER-RNA, ANDMETHIONYL-TRANSFER-RNA SYNTHETASE IN THE MACROMOLECULAR ARS COMPLEX

Rat liver cytosol was incubated with a trace amount of rat liver 5SrRN A which was highly labeled at the 3'-end with cytidine 3',5'-[5'-P-32] bisphosphate, and with [S-35]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 resu lts were obtained. (i) The eluate with glycine-HCl buffer (pH 3.0) fro m the protein A-Sepharose column contained an overlapping peak of P-32 - and S-35-radioactivities. In a control experiment using the same amo unt of P-32-labeled Escherichia coli 5SrRNA with the same specific act ivity, no fraction of the eluate contained P-32-radioactivity. (ii) Th e fractions containing both P-32- and S-35-radioactivities from the pr otein A-Sepharose column were crosslinked by UV irradiation. The produ cts was subjected to PAGE, and RNA in each gel slice was eluted and pu rified. The fraction containing both P-32- and S-35-radioactivities wa s present in a region of somewhat higher molecular weight than that of 5SRNP, whereas very low P-32- and S-35-radioactivities were present i n this region in the control experiment without UV irradiation, This f inding suggested that [S-35]methionyl-tRNA interacted with P-32-labele d 5SRNP. (iii) The fraction containing overlapping P-32- and S-35-radi oactivities described above was subjected to Sephadex G-150 chromatogr aphy. The component containing both radioactivities was distributed in the region corresponding to molecular weights of 10,000 to 250,000 wi th a peak at about 200,000, suggesting the presence of a complex conta ining Met-RS (M(r) 108,000), 5SRNP (M(r) 74,000), and methionyl-tRNA ( M(r) 25,000). Furthermore, this fraction showed definite Met-RS activi ty. These results indicate that P-32-5SRNP, met-tRNA, and Met-RS form a complex which may be released from the macromolecular ARS complex du ring the preparation procedures described above, Formation of this com plex may be the mechanism by which 5SrRNA(P) enhances the activity of Met-RS in the macromolecular ARS complex.