Mutations to kirromycin resistance occur in the interface of domains I and III of EF‐Tu·GTP

Abdulkarim, Farhad; Liljas, Lars; Hughes, Diarmaid

doi:10.1016/0014-5793(94)00937-6

Cited by 60 publications

(51 citation statements)

References 27 publications

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“…The kirromycin binding site is located at the interface between domains I and III of EF-Tu (12) (see Fig. S1 in the supplemental material), and previous studies have identified Kir r mutations at this interface (28). These observations are consistent with the proposal that domains I and III are dynamic with respect to one another (29).…”

Section: Resultssupporting

confidence: 84%

Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus

et al. 2015

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The bacterial ribosome and its associated translation factors are frequent targets of antibiotics, and antibiotic resistance mutations have been found in a number of these components. Such mutations can potentially interact with one another in unpredictable ways, including the phenotypic suppression of one mutation by another. These phenotypic interactions can provide evidence of long-range functional interactions throughout the ribosome and its functional complexes and potentially give insights into antibiotic resistance mechanisms. In this study, we used genetics and experimental evolution of the thermophilic bacterium Thermus thermophilus to examine the ability of mutations in various components of the protein synthesis apparatus to suppress the streptomycin resistance phenotypes of mutations in ribosomal protein S12, specifically those located distant from the streptomycin binding site. With genetic selections and strain constructions, we identified suppressor mutations in EF-Tu or in ribosomal protein L11. Using experimental evolution, we identified amino acid substitutions in EF-Tu or in ribosomal proteins S4, S5, L14, or L19, some of which were found to also relieve streptomycin resistance. The wide dispersal of these mutations is consistent with long-range functional interactions among components of the translational machinery and indicates that streptomycin resistance can result from the modulation of long-range conformational signals. IMPORTANCEThe thermophilic bacterium Thermus thermophilus has become a model system for high-resolution structural studies of macromolecular complexes, such as the ribosome, while its natural competence for transformation facilitates genetic approaches. Genetic studies of T. thermophilus ribosomes can take advantage of existing high-resolution crystallographic information to allow a structural interpretation of phenotypic interactions among mutations. Using a combination of genetic selections, strain constructions, and experimental evolution, we find that certain mutations in the translation apparatus can suppress the phenotype of certain antibiotic resistance mutations. Suppression of resistance can occur by mutations located distant in the ribosome or in a translation factor. These observations suggest the existence of long-range conformational signals in the translating ribosome, particularly during the decoding of mRNA. The ribosome is the highly conserved RNA-protein complex responsible for protein synthesis in all species and is the target of numerous antibiotics that bind to spatially dispersed, highly conserved functional sites (reviewed in references 1-3). Mutations arising in antibiotic binding sites often confer resistance, rendering such drugs ineffective. Given that the ribosome has multiple antibiotic binding sites, it is perhaps not surprising that mutations conferring resistance to one antibiotic can cause increased sensitivity to another. For instance, a C1066U base substitution in Escherichia coli 16S rRNA conferring spectinomycin resistance caus...

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Section: Resultssupporting

confidence: 84%

Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus

et al. 2015

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“…Substitution mutations at three amino acid sites in domain I of EF-Tu, Leu 12°, Gln 124, TyP 6° give rise to a kirromycin resistant phenotype [13]. The side chains of these three amino acids are aligned and in contact, Gln with Leu and Leu with Tyr, and all point into the domain I-III interface in both the GTP and GDP conformations of EF-Tu [1,31].…”

Section: Discussionmentioning

confidence: 99%

“…The basis of the causal relationship between kirromycin resistance and reduced affinity for aa-tRNA may be the location of the mutations in the domain interface, a location which could plausibly affect the conformational switching of EF-Tu, reducing its affinity for both ligands. This interpretation would suggest that though mutations to kirromycin resistance map to both sides of the domain I-III interface [13], the actual binding site of kirromycin might be elsewhere, for example on domain I, but be influenced by the interdomain interactions.…”

Section: Discussionmentioning

confidence: 99%

“…Residues in EF-I~ equivalent to residues (260-263) in domain II of E. coli EF-Tu are crosslinked to, and protected from protease digestion by, aa-tRNA [12]. The kirromycin resistant tufAa allele in E. coli, Glu3ZSLys in domain III of EF-Tu, is defective in binding aa-tRNA [5,13]. Truncated EF-Tu, lacking either domain I, or both domains II and III, shows no interaction with aa-tRNA [14,15].…”

Section: Introductionmentioning

confidence: 99%

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Mutants of EF‐Tu defective in binding aminoacyl‐tRNA

1996

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Five single amino acid substitution variants of EF-Tu from Salmonella typhimurlum were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leut2°Gln, Gln124Arg and Tyr 16° (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu. GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, kcat/KM for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu12°Gln, Gln124Arg and Tyrl6°Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu" GTP and aminoacyi-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124Arg change, where the association constant is 30-fold lower than in the wild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (ka) for the release of GDP from EF-Tu is increased 6-fold by the Tyrl6°Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

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“…Resistance to kirromycin is usually achieved by single amino acid substitutions in highly conserved positions of EFTu. These mutations cluster in or near the interface between domains I and III of EF-Tu⅐GTP (1,19), a region where the antibiotic was recently found to bind (38). Superposition of the EF-Tu2 amino acid sequence on the crystal structure of Thermus thermophilus EF-Tu⅐GppNHp (3) revealed that no deviating residues are present at the interface between domains I and III, although kirromycin resistance due to residues elsewhere in the protein cannot be excluded.…”

Section: Vol 184 2002 S Ramocissimus Tuf2 Encodes a Minor Ef-tu 4215mentioning

confidence: 99%

The Unique tuf2 Gene from the Kirromycin Producer Streptomyces ramocissimus Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu

2002

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Streptomyces ramocissimus, the producer of elongation factor Tu (EF-Tu)-targeted antibiotic kirromycin, contains three divergent tuf-like genes, with tuf1 encoding regular kirromycin-sensitive EF-Tu1; the functions of tuf2 and tuf3 are unknown. Analysis of the tuf gene organization in nine producers of kirromycin-type antibiotics revealed that they all contain homologues of tuf1 and sometimes of tuf3 but that tuf2 was found in S. ramocissimus only. The tuf2-flanking regions were sequenced, and the two tuf2-surrounding open reading frames were shown to be oriented in opposite directions. In vivo transcription analysis of the tuf2 gene displayed an upstream region with bidirectional promoter activity. The transcription start site of tuf2 was located approximately 290 nucleotides upstream of the coding sequence. Very small amounts of tuf2 transcripts were detected in both liquid-and surface-grown cultures of S. ramocissimus, consistent with the apparent absence of EF-Tu2 in total protein extracts. The tuf2 transcript level was not influenced by the addition of kirromycin to exponentially growing cultures. To assess the function of S. ramocissimus EF-Tu2, the protein was overexpressed in Streptomyces coelicolor LT2. This strain is a J1501 derivative containing His 6 -tagged EF-Tu1 as the sole EF-Tu species, which facilitated the separation of EF-Tu2 from the interfering EF-Tu1. S. ramocissimus EF-Tu1 and EF-Tu2 were indistinguishable in their ability to stimulate protein synthesis in vitro and exhibited the same kirromycin sensitivity, which excludes the possibility that EF-Tu2 is directly involved in the kirromycin resistance mechanism of S. ramocissimus.

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Mutations to kirromycin resistance occur in the interface of domains I and III of EF‐Tu·GTP

Cited by 60 publications

References 27 publications

Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus

Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus

Mutants of EF‐Tu defective in binding aminoacyl‐tRNA

The Unique tuf2 Gene from the Kirromycin Producer Streptomyces ramocissimus Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu

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