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- Mitkevich, Vladimir A., et al.
(författare)
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Thermodynamic Characterization of ppGpp Binding to EF-G or 1F2 and of Initiator tRNA Binding to Free 1F2 in the Presence of GDP, GTP, or ppGpp
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 402:5, s. 838-846
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Tidskriftsartikel (refereegranskat)abstract
- In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 degrees C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 Q mu M K-d versus 9.1-13.9 mu M K-d at 10-25 degrees C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA(fMet) demonstrating that IF2 binds to initiator tRNA with submicromolar K-d and that affinity is altered by the G nucleotides only slightly. This-in conjunction with earlier reports on IF2 interactions with fMet-tRNA(fMet) in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA(fMet) binding and GTP was suggested to strongly promote fMet-tRNA(fMet) binding-sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA(fMet) selection.
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| 2. |
- Murina, Victoriia, et al.
(författare)
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ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance : Structural and Functional Diversification across the Tree of Life
- 2019
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 431:18, s. 3568-3590
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Tidskriftsartikel (refereegranskat)abstract
- Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.
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