Yeast ribosomal protein deletion mutants possess altered peptidyltransferase activity and different sensitivity to cycloheximide

The major function of the ribosome is its ability to catalyze formation of peptide bonds, and it is carried out by the ribosomal peptidyltransferase. Recent evidence suggests that the catalyst of peptide bond formation is the 23S rRNA of the large ribosomal subunit. We have developed an in vitro sys tem for the determination of peptidyltransferase activity in yeast ribosome s. Using this system, a kinetic analysis of a model reaction for peptidyltr ansferase is described with Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The Ac-Phe-tRNA-poly(U)-80S ribosome complex (complex C) was isolated and then reacted with excess puromycin to give Ac-Phe-puromyci n. This reaction (puromycin reaction) followed first-order kinetics. At sat urating concentrations of puromycin, the first-order rate constant (k(3)) i s identical to the catalytic rate constant (k(cat)) of peptidyltransferase. This k(cat) from wild-type yeast strains was equal to 2.18 min(-1) at 30 d egreesC. We now present for the first time kinetic evidence that yeast ribo somes lacking a particular protein of the 60S subunit may possess significa ntly altered peptide bond-forming ability. The k(cat) of peptidyltransferas e from mutants lacking ribosomal protein L24 was decreased 3-fold to 0.69 m in(-1), whereas the k(cat) from mutants lacking L39 was slightly increased to 3.05 min(-1) and that from mutants lacking both proteins was 1.07 min(-1 ). These results suggest that the presence of ribosomal proteins L24 and, t o a lesser extent, L39 is required for exhibition of the normal catalytic a ctivity of the ribosome, Finally, the L24 or L39 mutants did not affect the rate or the extent of the translocation phase of protein synthesis. Howeve r, the absence of L24 caused increased resistance to cycloheximide, a trans location inhibitor. Translocation of Ac-Phe-tRNA from the A- to P-site was inhibited by 50% at 1.4 muM cycloheximide for the L24 mutant compared to 0. 7 muM for the wild type.