Download Interaction of cycloheximide with 25S ribosomal RNA from yeast

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.