| 1. |
- Andreev, Dmitri, et al.
(författare)
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The bacterial toxin ReIE induces specific mRNA cleavage in the A site of the eukaryote ribosome
- 2008
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Ingår i: RNA. - : RNA Society. - 1355-8382 .- 1469-9001. ; 14:2, s. 233-239
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Tidskriftsartikel (refereegranskat)abstract
- ReIE/ReIB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. ReIE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. ReIE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "ReIE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.
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| 2. |
- Borg, Anneli, et al.
(författare)
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Complete kinetic mechanism for recycling of the bacterial ribosome
- 2016
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 22:1, s. 10-21
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Tidskriftsartikel (refereegranskat)abstract
- How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of 5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.
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| 3. |
- Ieong, Ka-Weng, et al.
(författare)
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A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids
- 2014
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 20:5, s. 632-643
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Tidskriftsartikel (refereegranskat)abstract
- There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNAAla-based body (tRNAAlaB) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNAPheB body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNAAlaB body than from the tRNAPheB body. At ∼1 µM EF-Tu, tRNAAlaB conferred considerably faster incorporation kinetics than tRNAPheB, especially in the case of the bulky bK. In contrast, the swap to the tRNAAlaB body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNAAlaB and tRNAPheB bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.
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| 4. |
- Ivanova, Natalia, et al.
(författare)
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Structure probing of tmRNA in distinct stages of trans-translation
- 2007
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 13:5, s. 713-722
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was d dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.
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| 5. |
- Zhang, Jingji, et al.
(författare)
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Proofreading neutralizes potential error hotspots in genetic code translation by transfer RNAs
- 2016
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 22:6, s. 896-904
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Tidskriftsartikel (refereegranskat)abstract
- The ribosome uses initial and proofreading selection of aminoacyl-tRNAs for accurate protein synthesis. Anomalously high initial misreading in vitro of near-cognate codons by tRNAHis and tRNAGlu suggested potential error hotspots in protein synthesis, but in vivo data suggested their partial neutralization. To clarify the role of proofreading in this error reduction, we varied the Mg2+ ion concentration to calibrate the total accuracy of our cell-free system to that in the living Escherichia coli cell. We found the total accuracy of tRNA selection in our system to vary by five orders of magnitude depending on tRNA identity, type of mismatch, and mismatched codon position. Proofreading and initial selection were positively correlated at high, but uncorrelated at low initial selection, suggesting hyperactivated proofreading as a means to neutralize potentially disastrous initial selection errors.
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