Thiostrepton-resistant mutants of Thermus thermophilus.
Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic.
Cameron
Dale M
DM
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Thompson
Jill
J
Gregory
Steven T
ST
March
Paul E
PE
Dahlberg
Albert E
AE
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002455:Cell Division; D024881:Drug Resistance, Bacterial; D005785:Gene Conversion; D020459:Genes, rRNA; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D020653:Peptide Elongation Factor G / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000166:cytology / Q000187:drug effects* / Q000235:genetics*; D013883:Thiostrepton / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D020653:Peptide Elongation Factor G; D012335:RNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D006160:Guanosine Triphosphate; D013883:Thiostrepton