| 1. |
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| 2. |
- Choi, Junhong, et al.
(författare)
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N-6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics
- 2016
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 23:2, s. 110-
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Tidskriftsartikel (refereegranskat)abstract
- N-6-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.
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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. |
- Ieong, Ka-Weng, et al.
(författare)
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Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134:43, s. 17955-17962
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Tidskriftsartikel (refereegranskat)abstract
- Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural l-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA Phe. The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA Phe derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA Phe. We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.
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| 5. |
- Ieong, Ka-Weng, 1985-, et al.
(författare)
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N-6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:5, s. 2684-2699
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Tidskriftsartikel (refereegranskat)abstract
- We used quench flow to study how N-6-methylated adenosines (m(6)A) affect the accuracy ratio between k(cat)/K-m (i.e. association rate constant (k(a)) times probability (P-p) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated k(cat)/K-m for Glu-tRNA(Glu), EF-Tu and GTP forming ternary complex (T-3) reading cognate (GAA and Gm(6)AA) or near-cognate (GAU and Gm(6)AU) codons. k(a) decreased 10-fold by m(6)A introduction in cognate and near-cognate cases alike, while P-p for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated k(cat)/K-m for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um(6)AA) and near-cognate (UAG and Um(6)AG) stop codons to decrease 6-fold or 3-fold by m(6)A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m(6)A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate k(cat)/K-m, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate k(cat)/K-m.
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| 6. |
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| 7. |
- Ieong, Ka-Weng, 1985-
(författare)
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Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids?Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition.By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches.We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis.Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.
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| 8. |
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| 9. |
- Ieong, Ka-Weng, 1985-, et al.
(författare)
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Two proofreading steps amplify the accuracy of genetic code translation
- 2016
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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. ; 13:48, s. 13744-13749
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Tidskriftsartikel (refereegranskat)abstract
- Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aatRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EFTu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.
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| 10. |
- Johansson, Magnus, et al.
(författare)
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pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
- 2011
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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. ; 108:1, s. 79-84
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Tidskriftsartikel (refereegranskat)abstract
- We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.
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| 11. |
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| 12. |
- Volkov, Ivan, et al.
(författare)
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tRNA tracking for direct measurements of protein synthesis kinetics in live cells
- 2018
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Ingår i: Nature Chemical Biology. - : NATURE PUBLISHING GROUP. - 1552-4450 .- 1552-4469. ; 14:6, s. 618-626
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Tidskriftsartikel (refereegranskat)abstract
- Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy.Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA(Phe) that are in perfect agreement with previous indirect estimates, and once fMet-tRNA(fMet) has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.
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| 13. |
- Zhang, Jingji, et al.
(författare)
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Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome
- 2015
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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. ; 112:31, s. 9602-9607
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Tidskriftsartikel (refereegranskat)abstract
- We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.
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| 14. |
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| 15. |
- 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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