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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. |
- Aguirre Rivera, Javier, 1989-
(författare)
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Tracking single molecules in uncharted territory : A single-molecule method to study kinetics in live bacteria
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.
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| 5. |
- Sabantsev, Anton, et al.
(författare)
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Spatiotemporally controlled generation of NTPs for single-molecule studies
- 2022
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Ingår i: Nature Chemical Biology. - : Springer Nature. - 1552-4450 .- 1552-4469. ; 18:10, s. 1144-
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Tidskriftsartikel (refereegranskat)abstract
- Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.
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| 6. |
- Seefeldt, A. Carolin, et al.
(författare)
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Direct measurements of erythromycin’s effect on protein synthesis kinetics in living bacterial cells
- 2021
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 433:10
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Tidskriftsartikel (refereegranskat)abstract
- Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA(Phe) on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA(fMet). By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA(Phe), underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.
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