PRESENCE AND ROLE OF THE 5SRRNA-L5 PROTEIN COMPLEX (5SRNP) IN THE THREONYL-TRANSFER-RNA AND HISTIDYL-TRANSFER-RNA SYNTHETASE COMPLEX IN RAT-LIVER CYTOSOL

A complex containing Thr-RS and His-RS was purified about 1000 to 2000 -fold from rat liver cytosol by successive column chromatographies on Sephadex G-200, Phenyl-Sepharose CL-4B, and tRNA-Sepharose. The ratio of the specific activity of Thr-RS and His-RS was relatively constant throughout the purification steps, suggesting that the two synthetases were co-purified as a complex. Chromatographic analyses of the tRNA-S epharose fraction by Sephadex G-150 column chromatography showed the p resence of a hybrid form of the Thr-RS monomer and the His-RS monomer in addition to dimer forms of both enzymes from the pattern of activit y of both enzymes. The monomer form of Thr-RS showed high activity com parable to the dimer form and the monomer form of His-RS showed defini te activity. An association form of Thr-RS and His-RS dimers was detec ted by Sephadex G-200 chromatography of rat liver cytosol. Northern bl ot analysis of RNA prepared from the tRNA-Sepharose fraction showed th e presence of 5SrRNA. Dot blot analysis of the tRNA-Sepharose fraction using an antibody against ribosomal protein L5, showed the presence o f ribosomal protein L5 in this fraction. These findings suggest the pr esence of a 5SRNA-L5 protein complex (5SRNP) in the Thr-RS and His-RS complex. 5SRNP enhanced the activity of Thr-RS in a freshly prepared t RNA-Sepharose fraction. It also enhanced the activity of the rat liver cytosol for the attachment of [H-3]threonine to endogenous tRNA. This activity was inhibited by an antibody against protein L5, and the inh ibition was reversed by addition of 5SRNP. These results indicate that 5SRNP plays a role as a positive effector of Thr-RS in the complex.