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| 2. |
- Prabhakar, Arjun, et al.
(författare)
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Dynamics of release factor recycling during translation termination in bacteria
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:11, s. 5774-5790
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Tidskriftsartikel (refereegranskat)abstract
- In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.
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| 3. |
- Antoun, Ayman, et al.
(författare)
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How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis
- 2006
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 23:2, s. 183-193
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Tidskriftsartikel (refereegranskat)abstract
- During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNA(fMet) bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.
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| 4. |
- Antoun, Ayman, et al.
(författare)
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How initiation factors tune the rate of initiation of protein synthesis in bacteria.
- 2006
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 25:11, s. 2539-50
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Tidskriftsartikel (refereegranskat)abstract
- The kinetics of initiator transfer RNA ( tRNA) interaction with the messenger RNA ( mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency ( k(cat)/K-M) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.
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| 5. |
- Antoun, Ayman, et al.
(författare)
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The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis
- 2003
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 22:20, s. 5593-5601
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Tidskriftsartikel (refereegranskat)abstract
- The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.
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| 6. |
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| 7. |
- Cheng, Kimberley, et al.
(författare)
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tmRNA-SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes
- 2010
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1047-8477 .- 1095-8657. ; 169:3, s. 342-348
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 A resolution structure of the tmRNA-SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA-SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.
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| 8. |
- 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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| 9. |
- Fange, David, et al.
(författare)
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Identification of enzyme inhibitory mechanisms from steady-state kinetics
- 2011
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Ingår i: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 93:9, s. 1623-1629
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Tidskriftsartikel (refereegranskat)abstract
- Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists. Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.
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