A base substitution within the GTPase-associated domain of mammalian 28 S ribosomal RNA causes high thiostrepton accessibility.
A molecular basis for the insensitivity of eukaryotic ribosomes to the antibiotic thiostrepton was investigated using synthetic 100-nucleotide-long fragments covering the GTPase domain of 23/28 S rRNA. Filter binding assay showed no detectable binding of the rat RNA to thiostrepton, but the binding capacity was markedly increased by base substitution of G1878 to A at the position corresponding to 1067 of Escherichia coli 23 S rRNA. The association constant (K alpha) for the rat A 1878 mutant was 0.60 x 10(6) M-1, which was comparable with that of the E. coli RNA (K alpha = 1.1 x 10(6) M-1). This suggests that the eukaryotic G 1878 participates in the resistance for thiostrepton. On the other hand, the RNA fragments of the two species had a similar binding capacity for E. coli ribosomal protein L11 and its mammalian homologue L12. Gel electrophoresis under a high ionic condition, however, revealed a difference between the two proteins. E. coli L11 formed stable complexes with both the E. coli RNA and the rat A 1878 mutant RNA in the presence of thiostrepton, while rat L12 failed to exhibit such complex formation. This suggests that the eukaryotic L12 protein may also be an element giving the resistance for thiostrepton. These results are discussed in terms of preserved three-dimensional conformation of the RNA backbone between prokaryotes and higher eukaryotes.
Department of Biochemistry, Niigata University School of Medicine, Japan.
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Uchiumi
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Wada
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Kominami
D000241:Adenosine; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D006147:Guanine; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D012270:Ribosomes / Q000378:metabolism*; D013883:Thiostrepton / Q000378:metabolism*
D046911:Macromolecular Substances; D012329:RNA, Bacterial; D012339:RNA, Ribosomal, 28S; D006147:Guanine; D020558:GTP Phosphohydrolases; D013883:Thiostrepton; D000241:Adenosine