The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

B T Porse; I Leviev; A S Mankin; R A Garrett

doi:10.1006/jmbi.1997.1541

The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

B T Porse, I Leviev, A S Mankin, R A Garrett

98 Citations (Scopus)

Abstract

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.

Original language	English
Journal	Journal of Molecular Biology
Volume	276
Issue number	2
Pages (from-to)	391-404
Number of pages	13
ISSN	0022-2836
DOIs	https://doi.org/10.1006/jmbi.1997.1541
Publication status	Published - 1998

Access to Document

10.1006/jmbi.1997.1541

Cite this

@article{9128610074ca11dbbee902004c4f4f50,

title = "The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre",

abstract = "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.",

author = "Porse, {B T} and I Leviev and Mankin, {A S} and Garrett, {R A}",

note = "Keywords: Amino Acid Sequence; Anti-Bacterial Agents; DNA Footprinting; Drug Resistance, Microbial; GTP Phosphohydrolases; Halobacterium salinarum; Molecular Sequence Data; Mutation; RNA; RNA, Ribosomal, 23S; Recombinant Fusion Proteins; Ribosomal Proteins; Ribosomes; Thiostrepton",

year = "1998",

doi = "10.1006/jmbi.1997.1541",

language = "English",

volume = "276",

pages = "391--404",

journal = "Journal of Molecular Biology",

issn = "0022-2836",

publisher = "Academic Press",

number = "2",

}

TY - JOUR

T1 - The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

AU - Porse, B T

AU - Leviev, I

AU - Mankin, A S

AU - Garrett, R A

N1 - Keywords: Amino Acid Sequence; Anti-Bacterial Agents; DNA Footprinting; Drug Resistance, Microbial; GTP Phosphohydrolases; Halobacterium salinarum; Molecular Sequence Data; Mutation; RNA; RNA, Ribosomal, 23S; Recombinant Fusion Proteins; Ribosomal Proteins; Ribosomes; Thiostrepton

PY - 1998

Y1 - 1998

N2 - 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.

AB - 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.

U2 - 10.1006/jmbi.1997.1541

DO - 10.1006/jmbi.1997.1541

M3 - Journal article

C2 - 9512711

SN - 0022-2836

VL - 276

SP - 391

EP - 404

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 2

ER -

The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

Abstract

Access to Document

Fingerprint

Cite this