Interaction of 5SrRNA-L5 Protein Complex, Methionyl-tRNA, and Methionyl-tRNA Synthetase in the Macromolecular ARS Complex

抄録

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] bisphosphate, 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 (M_r 108, 000), 5SRNP (M_r 74, 000), and methionyl-tRNA (M_r 25, 000). Furthermore, this fraction showed definite Met-RS activity. These results indicate that ³²P-5SRNP, met-tRNA, and Met-RS form a complex which may be released from the macromolecular ARS complex during the preparation procedures described above. Formation of this complex may be the mechanism by which 5SrRNA(P) enhances the activity of Met-RS in the macromolecular ARS complex.