The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre.
A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.
Porse
B T
BT
RNA Regulation Centre, University of Copenhagen, Denmark.
Leviev
I
I
Mankin
A S
AS
Garrett
R A
RA
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018983:DNA Footprinting; D004352:Drug Resistance, Microbial / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000187:drug effects* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects; D013883:Thiostrepton / Q000378:metabolism / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D012338:RNA, Ribosomal, 23S; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D012313:RNA; D020558:GTP Phosphohydrolases; D013883:Thiostrepton