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Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

ABSTRACT: Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.

SUBMITTER: Olivier NB

PROVIDER: S-EPMC4246262 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Olivier Nelson B NB Altman Roger B RB Noeske Jonas J Basarab Gregory S GS Code Erin E Ferguson Andrew D AD Gao Ning N Huang Jian J Juette Manuel F MF Livchak Stephania S Miller Matthew D MD Prince D Bryan DB Cate Jamie H D JH Buurman Ed T ET Blanchard Scott C SC

Proceedings of the National Academy of Sciences of the United States of America 20141103 46

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with t ...[more]

PMID: 25368144

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Project description:BackgroundEscherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations.ResultsProduction of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg2+, indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising.ConclusionsThrough characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production.

Dataset Information

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Publications

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

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