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
Autor: | Bo T. Porse, Mariana J. Awayez, Harry C. J. Ottenheijm, S.V. Kirillov, Roger A. Garrett |
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Rok vydání: | 1999 |
Předmět: |
Halobacterium salinarum
Peptidyl transferase Molecular Sequence Data Saccharomyces cerevisiae macromolecular substances Ribosome chemistry.chemical_compound RNA Transfer 23S ribosomal RNA Escherichia coli Ultraviolet light Humans Peptide bond Antibiotics Antineoplastic Multidisciplinary Base Sequence biology technology industry and agriculture RNA Fungal Sparsomycin Biological Sciences Ribosomal RNA RNA Bacterial RNA Ribosomal 23S Cross-Linking Reagents chemistry Biochemistry Peptidyl Transferases Transfer RNA Bacillus megaterium biology.protein Nucleic Acid Conformation Ribosomes |
Zdroj: | Proceedings of the National Academy of Sciences. 96:9003-9008 |
ISSN: | 1091-6490 0027-8424 |
DOI: | 10.1073/pnas.96.16.9003 |
Popis: | 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. |
Databáze: | OpenAIRE |
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