| 1. |
- Abdalaal, Hind, et al.
(författare)
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Collateral toxicity limits the evolution of bacterial Release Factor 2 towards total omnipotence
- 2020
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:10, s. 2918-2930
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Tidskriftsartikel (refereegranskat)abstract
- When new genes evolve through modification of existing genes, there are often trade-offs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near cognate stop codons and highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds not only to alterations in the demand for RF2 activity, but also for RF1 activity.
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| 2. |
- Aguirre Rivera, Javier, 1989-, et al.
(författare)
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Real-time measurements of aminoglycoside effects on protein synthesis in live cells
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 118:9
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Tidskriftsartikel (refereegranskat)abstract
- The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.
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| 3. |
- Albers, Suki, et al.
(författare)
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Repurposing tRNAs for nonsense suppression
- 2021
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the T Psi C-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 angstrom resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon. Here, the authors report de novo design, optimization and characterization of tRNAs that decode UGA stop codons in E. coli. The structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon suggests that distinct A-site ligands (tRNAs versus release factors) induce distinct conformation of the stop codon within the mRNA in the decoding center.
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| 4. |
- Chen, Yu-Xiang, et al.
(författare)
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Selective translation by alternative bacterial ribosomes
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:32, s. 19487-19496
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Tidskriftsartikel (refereegranskat)abstract
- Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.
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| 5. |
- De Tarafder, Arindam
(författare)
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Adaptive Evolution of the Bacterial Translation Machinery
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The process of protein synthesis via translation is of paramount importance for the existence of life on Earth. The bacterial translation machinery has embraced more than 3.5 billion years of molecular evolution to adapt and function efficiently under the provided physiological conditions. This thesis dwells on the intricacies of the adaptive evolution, which the massively complex translation machinery has undergone to function optimally in diverse conditions and habitats. In Paper I, we used elongation factor Tu (EF-Tu) as a model system to follow the evolution of ribosome specificity in translation factors. For that, we have biochemically characterized two sequence-reconstructed ancestral EF-Tu variants for their specificities towards two unrelated extant bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. Our fast kinetics-based biochemical analysis hints at the ‘generalist’ ancestry of modern EF-Tu proteins. In Paper II, we have reconstituted an in vitro translation system of the psychrotolerant bacteria Pseudoalteromonas haloplanktis to quantitatively characterize the steps of translation elongation. Our results demonstrate similar kinetics of peptide bond formation in psychrotolerant P. haloplanktis and mesophilic E. coli. In contrast, P. haloplanktis ribosome exhibits much slower rates of EF-G-catalyzed tRNA translocation than E. coli. Comparison and swapping of the EF-Gs and tRNAs between the two in vitro translation systems indicate that the slow translocation is likely an inherent property of the P. haloplanktis ribosome. Furthermore, our results demonstrate the varied extent of antibiotic inhibition on the P. haloplanktis minimal translation system, particularly when targeting processes related to translocation and peptide bond formation, compared to E. coli. In Paper III, we used ribosomes from bacterial species of diverse habitats to show that the ribosomes in vitro can maintain their catalytic activity beyond the survival temperature cutoff of the native host. Moreover, our results indicate that the thermostability of essential translation factors, EF-Tu and EF-G, dictates the upper limit of reaction temperature for translation elongation. Finally, we demonstrate that ribosomes from a psychrophile, mesophile, and thermophile can function in a vast temperature range of 10-70 °C, provided the translation factors remain structurally and functionally stable. Our results highlight the thermal versatility of the ribosome and reiterate the early emergence of a thermostable ribosomal core in the primordial RNA world.The outcome of this thesis will unveil some of the intricate mechanisms underlying the evolution of bacterial translation machinery. This knowledge may open up new research avenues regarding the emergence and diversification of bacteria and the development of new therapeutic strategies.
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| 6. |
- De Tarafder, Arindam, et al.
(författare)
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Kinetic Analysis Suggests Evolution of Ribosome Snecificity in Modern Elongation Factor-Tus from "Generalist" Ancestors
- 2021
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Ingår i: Molecular biology and evolution. - : Oxford University Press. - 0737-4038 .- 1537-1719. ; 38:8, s. 3436-3444
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Tidskriftsartikel (refereegranskat)abstract
- It has been hypothesized that early enzymes are more promiscuous than their extant orthologs. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing presteady state fast kinetics using quench flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3- to 3.3-Gy-old ancestral EF-Tus toward two unrelated bacterial ribosomes, mesophilic Escherichia coil and thermophilic Thermus thermophilus. Although the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. In addition, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remains virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, "generalist" ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.
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| 7. |
- Eaglesfield, Ross, et al.
(författare)
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Cotranslational recruitment of ribosomes in protocells recreates a translocon-independent mechanism of proteorhodopsin biogenesis
- 2021
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Ingår i: iScience. - : Cell Press. - 2589-0042. ; 24:5
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of lipid membranes and embedded proteins was essential for the evolution of cells. Translocon complexesmediate cotranslational recruitment and membrane insertion of nascent proteins, but they already contain membrane-integral proteins. Therefore, a simpler mechanism must exist, enabling spontaneous membrane integration while preventing aggregation of unchaperoned protein in the aqueous phase. Here, we used giant unilamellar vesicles encapsulating minimal translation components to systematically interrogate the requirements for insertion of the model protein proteorhodopsin (PR) - a structurally ubiquitousmembrane protein. We show that the N-terminal hydrophobic domain of PR is both necessary and sufficient for cotranslational recruitment of ribosomes to the membrane and subsequent membrane insertion of PR. Insertion of N-terminally truncated PR was restored by artificially attaching ribosomes to the membrane. Our findings offer a self-sufficient protein-inherent mechanism as a possible explanation for effective membrane protein biogenesis in a "pre-translocon'' era, and they offer new opportunities for generating artificial cells.
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| 8. |
- Emmerich, Andrew
(författare)
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Cryo-EM and Computational Biology of Macromolecular Complexes
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ribosome is a large, ancient multicomponent macromolecular complex which is highly amenable to study by cryogenic electron microscopy (cryo-EM) and computation biology methods. This thesis delves into the structure of both prokaryotic and eukaryotic ribosomes in the context of determining a solution to emerging antimicrobial resistance. We show that thermorubin (THB) binds to the E. coli ribosome at intersubunit bridge B2a, flipping out 23S rRNA residue C1914 which interferes with A-site substrates. The position and rearrangements caused by THB also accounts for the biochemical results showing a decrease in elongation, termination and recycling phases of translation. Also using cryo-EM we looked at the Giardia intestinalis ribosome, determining six high-resolution structures representing translocation intermediates. Giardia is a protozoan parasite causing diarrhoea in humans, with metronidazole strains emerging. As the ribosome is often a target for antimicrobial drugs, work on the structure and function of the ribosome is of utmost important in determining an alternative therapeutic approach to the treatment of giardiasis. We also show naturally bound tRNAs and eEF2 on the Giardia ribosome, exhibiting eukaryote-specific subunit rolling and eEF2 with GDP in a uniquely positioned Pi primed for release, adding to the mechanism of translocation in protists as well as illustrating the evolution of both the structure and function of translation machinery. Finally, the molecular basis of thermostability in translational GTPases is explored using molecular dynamics of mesophilic and thermophilic elongation factor EF-Tu. Through ancestral sequence reconstruction two key interactions: in the GTPase domain; and an interdomain interaction were shown to be important in the overall structural stability of EF-Tu in high temperature environments. These studies together highlight the strength of utilising both structural and computational techniques to explore the translation apparatus.
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| 9. |
- Holm, Mikael, 1984-, et al.
(författare)
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Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
- 2024
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Tidskriftsartikel (refereegranskat)abstract
- Using pre-steady state kinetics and an E. coli based in vitro translation system we have studied the effect of the antibiotic viomycin on mRNA decoding. We find that viomycin binds to the ribosome during initial selection of tRNA, after binding of ternary complex but before GTP hydrolysis by EF-Tu. Viomycin binding renders the ribosome completely incapable of rejecting incorrect A-site bound tRNAs in both initial selection and proofreading. Viomycin sensitivity correlates with the accuracy of initial selection for the four different codon·anticodon pairs tested here. Our results demonstrate that, in contrast to current ideas about ‘induced-fit’, accuracy in initial selection is achieved primarily by increased dissociation rates for near-cognate tRNAs rather than by decreased rates of GTP hydrolysis. Further, our results imply that the ‘monitoring’ bases A1492 and A1493 rapidly fluctuate between active and inactive conformations when a near-cognate tRNA is present in the A site.
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| 10. |
- Kim, Changil, et al.
(författare)
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Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation
- 2021
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Ingår i: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 18:12, s. 2363-2375
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Tidskriftsartikel (refereegranskat)abstract
- Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNAPhe, with the natural dipeptidyl fMet-Phe-tRNAPhe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.
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