TY - JOUR
T1 - 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
AU - Porse, B T
AU - Kirillov, S V
AU - Awayez, M J
AU - Ottenheijm, H C
AU - Garrett, R A
PY - 1999/8/3
Y1 - 1999/8/3
N2 - 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.
AB - 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.
KW - Antibiotics, Antineoplastic/metabolism
KW - Bacillus megaterium/metabolism
KW - Base Sequence
KW - Cross-Linking Reagents/metabolism
KW - Escherichia coli/metabolism
KW - Halobacterium salinarum/metabolism
KW - Humans
KW - Molecular Sequence Data
KW - Nucleic Acid Conformation
KW - Peptidyl Transferases/chemistry
KW - RNA, Bacterial/metabolism
KW - RNA, Fungal/metabolism
KW - RNA, Ribosomal, 23S/chemistry
KW - RNA, Transfer/chemistry
KW - Ribosomes/drug effects
KW - Saccharomyces cerevisiae/metabolism
KW - Sparsomycin/analogs & derivatives
U2 - 10.1073/pnas.96.16.9003
DO - 10.1073/pnas.96.16.9003
M3 - Journal article
C2 - 10430885
SN - 0027-8424
VL - 96
SP - 9003
EP - 9008
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 16
ER -