| 1. |
- Fislage, Marcus, et al.
(författare)
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Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-Tu(H84A)
- 2018
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 46:11, s. 5861-5874
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Tidskriftsartikel (refereegranskat)abstract
- The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of themonitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.
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| 2. |
- Johansson, Magnus, et al.
(författare)
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Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection
- 2012
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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. ; 109:1, s. 131-136
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Tidskriftsartikel (refereegranskat)abstract
- Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg(2+) concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon-anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.
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| 3. |
- Krahn, Natalie, et al.
(författare)
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tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut
- 2024
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 52:2, s. 513-524
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Tidskriftsartikel (refereegranskat)abstract
- Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).
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| 4. |
- 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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| 5. |
- Prabhakar, Arjun, et al.
(författare)
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Uncovering translation roadblocks during the development of a synthetic tRNA
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:18, s. 10201-10211
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
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| 6. |
- Zhang, Jingji, et al.
(författare)
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Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions
- 2018
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 46:3, s. 1362-1374
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Tidskriftsartikel (refereegranskat)abstract
- We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T-3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T-3 from its initial codon-and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.
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| 7. |
- 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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| 8. |
- Zhang, Jingji, 1983-
(författare)
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Accuracy of mRNA Translation in Bacterial Protein Synthesis
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch).Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides. We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.
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| 9. |
- Zhang, Jingji, 1983-
(författare)
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Enhanced proofreading attenuates initial selection error hot spots in genetic code translation by transfer RNAs
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A system for cell free protein synthesis with E. coli components of high purity was used in conjunction with fast kinetics quench-flow measurements to characterize the accuracy of peptie bond formation by ribosomes with initiator tRNAfMet in P site and different codons in the A site. We used Glu-tRNAGlu, Lys-tRNALys and Phe-tRNAPhe in ternary complexes with EF-Tu and GTP to select ribosomes programmed with their respective cognate codons in competition with ribosomes containing near-cognate codons. Variation of the free Mg2+ concentration in the in vitro buffer system was used to calibrate its accuracy to that of codon selection by Glu-tRNAGlu in living E. coli cells, previously estimated from the residual activity of a beta-galactosidase mutant in which the codon for an essential Glu had been altered to near cognate codons. At 2.3 mM free Mg2+ concentration, the accuracy in the living cell agreed with that in the test tube, a feature making our biochemistry directly relevant to bacterial physiology. We found that the total accuracy of tRNA selection varied by five orders of magnitude depending on the type of tRNA, type of mismatch and mismatched codon position. We partitioned the total accuracy into initial selection of ternary complex before GTP hydrolysis on EF-Tu and proofreading selection of aminoacyl-tRNA after GTP hydrolysis. We found the contribution of proofreading to be strongly positively correlated with the accuracy of initial selection in its high range. As initial selection decreased further the proofreading contribution to accuracy increased, rather than decreased, a feature neutralizing potentially disastrous missense error hot spots associated with, in particular, tRNAGlu.
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| 10. |
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