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- 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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- 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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- 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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- 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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