The antitumor
antibiotic sparsomycin is a universal and potent inhibitor of
peptide bond formation and selectively acts on several human
tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor
tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between
sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound
tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved
nucleotide A2602 within the
peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach,
RNase H fragment analysis, and crosslinking with radioactive [(125)I]
phenol-alanine-sparsomycin. Crosslinking of several
sparsomycin derivatives, modified near the sulfoxy group, implicated the modified
uracil residue in the rRNA crosslink. The yield of the
antibiotic crosslink was weak in the presence of deacylated
tRNA and strong in the presence of an N-blocked P-site-bound
tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for
antibiotic inhibition. This supposition is reinforced by the observation that other
antibiotics that can prevent
peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink.