Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes.
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.
Porse
B T
BT
RNA Regulation Centre, Institute of Molecular Biology, University of Copenhagen, Solvgade 83H, DK1307 Copenhagen K, Denmark.
Kirillov
S V
SV
Awayez
M J
MJ
Ottenheijm
H C
HC
Garrett
R A
RA
D000903:Antibiotics, Antineoplastic / Q000378:metabolism* / Q000494:pharmacology; D001410:Bacillus megaterium / Q000378:metabolism; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010458:Peptidyl Transferases / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013033:Sparsomycin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology
D000903:Antibiotics, Antineoplastic; D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012338:RNA, Ribosomal, 23S; D013033:Sparsomycin; D012343:RNA, Transfer; D010458:Peptidyl Transferases