PARTIAL CHARACTERIZATION OF THE RIBONUCLEASE-P FROM TETRAHYMENA-PYRIFORMIS

Ribonuclease P activity from infusoria Tetrahymena pyriformis has been isolated and purified more than 1000-fold over cytosol crude extract. Purified tRNA 5' endonuclease processes in vitro heterologous substra tes, precursors of the human tRNA(Tyr) and Drosophila melanogaster tRN A(Leu), exactly at the 5' end of the mature molecules. The activity wa s abolished by micrococcal nuclease and protease treatment indicating that both RNA and protein components are essential for its activity. T he most abundant polypeptides in the purified enzyme fractions have mo lecular masses of about 100, 44 and 35 kDa. The enzyme requires divale nt cations for its activity and shows optimal activity in the presence of the low concentrations of the monovalent salts. Substrate structur al requirements for the purified enzyme were analyzed with different t RNA precursor models. The analysis of the derivatives of tRNA(Leu) pre cursors with altered aminoacyl stem structures reveals that end of the stem is important for substrate 5' end processing with purified enzym e. (C) Societe francaise de biochimie et biologie moleculaire / Elsevi er, Paris.