The binding site of puromycin was probed chemically
in the peptidyl-transferase center of ribosomes from Escherichia
coli and of puromycin-hypersensitive ribosomes from
the archaeon Haloferax gibbonsii. Several nucleotides
of the 23S rRNAs showed altered chemical reactivities in
the presence of puromycin. They include A2439, G2505, and
G2553 for E. coli, and G2058, A2503, G2505, and
G2553 for Hf. gibbonsii (using the E. coli
numbering system). Reproducible enhanced reactivities were
also observed at A508 and A1579 within domains I and III,
respectively, of E. coli 23S rRNA. In further
experiments, puromycin was shown to produce a major reduction
in the UV-induced crosslinking of deacylated-(2N3A76)tRNA
to U2506 within the P′ site of E. coli ribosomes.
Moreover, it strongly stimulated the putative UV-induced
crosslink between a streptogramin B drug and m2A2503/Ψ2504
at an adjacent site in E. coli 23S rRNA. These
data strongly support the concept that puromycin, along
with other peptidyl-transferase antibiotics, in particular
the streptogramin B drugs, bind to an RNA structural motif
that contains several conserved and accessible base moieties
of the peptidyl transferase loop region. This streptogramin
motif is also likely to provide binding sites for the 3′
termini of the acceptor and donor tRNAs. In contrast, the
effects at A508 and A1579, which are located at the exit
site of the peptide channel, are likely to be caused by
a structural effect transmitted along the peptide channel.