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Biochemical Society Transactions (1 994) 22 449s Interaction of cycloheximide with 255 ribosomal RNA from yeast. MICHAEL CANNON, M.AMIN A. MlRZA and PAUL R. BROWN. Molecular Biology and Biophysics Group, King's College London, Strand, London, WC2R 2LS. Cycloheximide is a glutarimide antibiotic that inhibits protein synthesis selectively on eukaryotic 80s ribosomes. The drug interferes with the translocation of peptidyl-tRNA from the acceptor to the donor site of ribosomes and its target site is located on the larger ribosomal subunit. Cycloheximide is thought to inhibit the functioning of the eukaryotic translocation factor EF-2. Ribosomes from the yeast Kluyveromyces lactis are completely resistant to inhibition by cycloheximide and this resistance is controlled by the K-lactis ribosomal protein L41 (Dehoux et al., 1993). Tn-GiEast, ribosomes from the yeast SZEFZromyces cerevisiae are inhibited by cycloheximide. This yeast possesses a ribosomal protein L41 that has a different primary structure from that of its counterpart in K. lact i s . The above observations suggest that the ribosomal domain that is, in S.cerevisiae, involved with the interaction of cyclEheximide contains ribosomal protein L41. It is likely, however. that the actual binding site for the drug is located on ribosomal RNA. RNA target sites have now been confirmed for a large number of antibiotics (see, for example, Moazed & Noller, 1987) and there is no reason to believe that cycloheximide is exceptional in this respect. It is reasonable to predict that the drug binds to a site on 25s ribosomal RNA that is associated, in ribosomes, with ribosomal protein L41. This possibility is explored in the present communication. Ribosomes were prepared from S.cerevisiae and were incubated with cycloheximide at a drug concentration that is known to inhibit protein synthesis in yeast cell-free systems. The ribosomes were then subjected to chemical modification, in both the absence and the presence of cycloheximide, using either dimethyl sulphate or kethoxal. The experimental conditions that were used were essentially as described previously (Woodcock et al., 1991). Dimethyl sulphate modifies adenine reFidiFs at the N1 position and cytosine residues at the N3 position. Kethoxal modifies guanine residues at both the N1 and N2 positions. it is predicted that the binding of cycloheximide to a specific site(s) on the 605 ribosomal subunit will protect that site(s) against chemical modification by dimethyl sulphate or by kethoxal. The sites of chemical modification and the drug protection site(s) are detected by isolating the ribosomal RNA and carrying out primer extension using suitable DNA oligomers that prime the action of the enzyme reverse transcriptase. After the above experiments had been carried out, sequencing gels revealed that cycloheximide protected two guanine residues against chemical modification. This result indicates strongly that ribosomes from the drug-sensitive S-cerevisiae strain have a.target site for cycloheximide that is located on 255 ribosomal RNA. By locating the position of the residues in published secondary structure models for yeast RNA it is clear that the protected residues lie within a domain that is closely adjacent to a conserved loop within the structure. This loop has been implicated in several functions of the ribosome that involve the hydrolysis of GTP. The ribosomal domain controls the interaction of the elongation factor EF-2, a protein that is involved in the translocation step of peptide bond formation. It should be noted, of course, that translocation is the reaction in protein synthesis that is thought to be inhibited by cycloheximide. It is also of interest that the conserved loop is, in euiaryotes, the site of attack by the RNA-directed cytotoxins alpha sarcin and ricin (see Moazed g g . , 1988). It is relevant to consider our results in the light of the fact that ribosomes from K.lactis are resistant to cycloheximide by virtue o f -possession of a particular form of ribosomal protein L41. Because our results indicate that cycloheximide has its target site on ribosomal RNA the implication is that the L41 protein in K.lactis affects the conformation of the ribosomal R N A m i s strain in such a way that the drug cannot bind. In contrast, in the drug-sensi t ive S-cereviside strains, the ribosomal protein L41 c o d i n e s with the relevant ribosomal RNA domain to form the cycloheximide receptor site. Despite these differences, however, our results support the idea that the ribosomal RNA of eukaryotes is likely to be the fundamental determinant in protein synthesis. We should like to acknowledge the financial support of the Wellcome Trust through a project grant. References Dehoux, P., Davies, J. & Cannon, M. (1993). Natural cycloheximide resistance in yeast: the role of ribosomal protein L41. Eur. J. Biochem. 213, 841-848. Moazed. D. & Noller, H.F. (1987). Interaction of antibiotics with functional sites in 165 ribosomal RNA. Nature, 327. 389-394. Moazed, D., Robertson, J.M. & Noller, H.F. (1988). Interaction of elongation factors EF-G and EF-TU with a conserved loop in 23s RNA. Nature, 334, 362364. WoodcocK. J., Moazed, D., Cannon, M., Davies. J. & Noller, H.F. (1991). Interaction of antibiotics with A- and P-site-specific bases in 165 ribosomal RNA. EMBO J. lo, 3099-3103.