A structural basis for streptomycin-induced misreading of the genetic code

DeMi̇rci̇, Hasan; Murphy, F.V.; Murphy, E.; Gregory, Steven T.; Dahĺberg, Albert E.; Jogl, G.

doi:10.1038/ncomms2346

Cited by 121 publications

(149 citation statements)

References 44 publications

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“…In contrast, ethanol did not show cooperative synergy with chloramphenicol, which inhibits peptidyl transfer in the ribosome but does not decrease translational accuracy (37), suggesting that ethanol does not impede the peptidyl transfer reaction. These results strongly indicate that sublethal ethanol stress induces physiologically damaging levels of translational misreading, perhaps compromising the conformation of the ribosomal decoding site known to be targeted by streptomycin (38,39).…”

Section: Resultsmentioning

confidence: 80%

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Haft

Keating

Schwaegler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

121

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The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

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Section: Resultsmentioning

confidence: 80%

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Haft

Keating

Schwaegler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

121

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“…The inherent plasticity of the decoding site is evident from structural investigations of decoding-site oligonucleotide analogs (Fourmy et al 1996(Fourmy et al , 1998, ribosomes in cognate decoding complexes (Ogle et al 2001;Schmeing et al 2009), and ribosomes in complexes with antibiotics Borovinskaya et al 2008;Stanley et al 2010;Demirci et al 2013) or initiation factor IF1 . The effects of SmD mutations on global aspects of ribosome conformation have been revealed by chemical probing (Allen and Noller 1989) and crystallography (Vila-Sanjurjo et al 2003), but the mechanistic underpinnings of the SmD phenotype remain unclear.…”

Section: Introductionmentioning

confidence: 99%

The central role of protein S12 in organizing the structure of the decoding site of the ribosome

DeMi̇rci̇

Wang

Murphy

et al. 2013

RNA

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The ribosome decodes mRNA by monitoring the geometry of codon-anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EFTu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.

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“…Several biochemical and structural studies have demonstrated that this miscoding occurs at the step of ternary complex (EF-Tu-GTP-amino acid tRNA) binding to the ribosomal A site (2). In the presence of Str, the forward rate constant of the next kinetic step, GTPase activation and hydrolysis, is indistinguishable for both cognate or near-cognate ternary complexes, resulting in reduced accuracy of amino acid-tRNA selection (2)(3)(4).…”

mentioning

confidence: 99%

Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

Chen

Blumentritt

Curtis

et al. 2013

Antimicrob Agents Chemother

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dStreptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Str r ) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Str r mutants of Escherichia coli O157:H7 have been generated for in vivo studies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) of E. coli O157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB in E. coli O157:H7 Str r restrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions in E. coli O157:H7. Therefore, we suggest that E. coli O157:H7 strains selected for Str r should be thoroughly characterized before in vivo and in vitro experiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.

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A structural basis for streptomycin-induced misreading of the genetic code

Cited by 121 publications

References 44 publications

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

The central role of protein S12 in organizing the structure of the decoding site of the ribosome

Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

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