| 1. |
- Choi, Junhong, et al.
(författare)
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2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation
- 2018
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Ingår i: Nature Structural & Molecular Biology. - : Springer Nature. - 1545-9993 .- 1545-9985. ; 25:3, s. 208-216
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Tidskriftsartikel (refereegranskat)abstract
- Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2 '-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2 '-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2 '-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.
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| 2. |
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| 3. |
- Kwiatkowski, Marek, et al.
(författare)
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Facile Synthesis of N-Acyl-aminoacyl-pCpA for Preparation of Mischarged Fully Ribo tRNA
- 2014
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 25:11, s. 2086-2091
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Tidskriftsartikel (refereegranskat)abstract
- Chemical synthesis of N-acyl-aminoacyl-pdCpA and its ligation to tRNA(minus) CA is widely used for the preparation of unnatural aminoacyl-tRNA substrates for ribosomal translation. However, the presence of the unnatural deoxyribose can decrease incorporation yield in translation and there is no straightforward method for chemical synthesis of the natural ribo version. Here, we show that pCpA is surprisingly stable to treatment with strong organic bases provided that anhydrous conditions are used. This allowed development of a facile method for chemical aminoacylation of pCpA. Preparative synthesis of pCpA was also simplified by using t-butyl-dithiomethyl protecting group methodology, and a more reliable pCpA postpurification treatment method was developed. Such aminoacyl-pCpA analogues ligated to tRNA(minus) CA transcripts are highly active in a purified translation system, demonstrating utility of our synthetic method.
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| 4. |
- Liljeruhm, Josefine, et al.
(författare)
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Kinetics of D-amino acid incorporation in translation
- 2019
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 14:2, s. 204-213
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Tidskriftsartikel (refereegranskat)abstract
- Despite the stereospecificity of translation for l-amino acids (l-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of d-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting d-AA incorporation need clarification. In this work, we compared d- and l-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the d isomer from a tRNAPhe-based adaptor (tRNAPheB). Incorporation was moderately hastened by tRNA body swaps and higher EF-Tu concentrations, indicating that binding by EF-Tu can be rate-limiting. However, from tRNAAlaB with a saturating concentration of EF-Tu, the slow d-Phe incorporation was unexpectedly very efficient in competition with incorporation of the l isomer, indicating fast binding to EF-Tu, fast binding of the resulting complex to the ribosome, and rate-limiting accommodation/peptide bond formation. Subsequent elongation with an l-AA was confirmed to be very slow and inefficient. This understanding helps rationalize incorporation efficiencies in vitro and stereospecific mechanisms in vivo and suggests approaches for improving incorporation.
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| 5. |
- Liljeruhm, Josefine, et al.
(författare)
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Plasticity and conditional essentiality of modification enzymes for domain V of Escherichia coli 23S ribosomal RNA
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 28:6, s. 796-807
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2 ' O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt "critical region" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20 degrees C): RlmE and RluC. At a permissive temperature (37 degrees C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37 degrees C, major at 20 degrees C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37 degrees C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.
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| 6. |
- 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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| 7. |
- Shepherd, Tyson R, et al.
(författare)
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De novo design and synthesis of a 30-cistron translation-factor module
- 2017
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 45:18, s. 10895-10905
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Tidskriftsartikel (refereegranskat)abstract
- Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.
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| 8. |
- Wang, Jinfan, 1989-
(författare)
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In Vitro Kinetics of Ribosomal Incorporation of Unnatural Amino Acids
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribosomal incorporation of unnatural amino acids (AAs) into peptides or proteins has found broad applications in studying translation mechanism, discovering potential therapeutics, and probing protein structure and function. However, such applications are generally limited by the low incorporation efficiencies of the unnatural AAs.With in vitro kinetics studies using a purified E. coli translation system, we found that the natural N-alkyl AA carrier, tRNAPro, could hasten the incorporation of N-methyl AAs. Also, the incorporation rate increased remarkably with increasing pH in the range of 7 to 8.5, suggesting the rate was limited by peptidyl transfer, not accommodation. Competition experiments revealed that several futile cycles of delivery and rejection of the A site N-methyl AA-tRNA were required per peptide bond formation, and the incorporation yield could be increased by using a higher Mg2+ concentration.Kinetics of ribosomal polymerization, using AA-tRNA substrates prepared from the standard N-NVOC-AA-pdCpA chemoenzymatic ligation method, clarified that the inefficiency of incorporation was due to the penultimate dC. This dC prompted faster peptidyl-tRNA drop-off, leading to loss of processivities along consecutive incorporations. Circumventing the penultimate dC by using our N-NVOC-AA-pCpA chemoenzymatic ligation or the flexizyme charging method to prepare the AA-tRNA substrates was able to improve the efficiencies of ribosomal consecutive incorporations of unnatural AAs.By studying the translation steps after aminoacylation of tRNAPyl, the favored carrier for unnatural AAs in vivo, we demonstrated surprisingly slow biphasic kinetics of tRNAPyl-mediated amber suppression in vitro. The fast phase amplitude increased with increasing EF-Tu concentration, allowing measurement of Kd of EF-Tu binding. Results revealed ~25-fold weaker EF-Tu binding affinity of the tRNAPyl body than that of E. coli tRNAPhe. The fast phase rate was ~30-fold slower than that of native substrates, and this rate was limited by the ~10-fold less efficient AA-tRNAPyl:EF-Tu:GTP ternary complex binding to the ribosome. The incorporation was so slow that termination by RF2 mis-reading of the amber codon became a significant competing reaction. The processivity was unexpectedly impaired as ~40% of the dipeptidyl-tRNAPyl could not be elongated to tripeptide. This new overall understanding opens a window of improving unnatural AA incorporation both in vitro and in vivo.
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| 9. |
- Wang, Jinfan, et al.
(författare)
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Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements
- 2015
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:10, s. 2187-2192
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomal synthesis of polymers of unnatural amino acids (AAs) is limited by low incorporation efficiencies using the artificial AA-tRNAs, but the kinetics have yet to be studied. Here, kinetics were performed on five consecutive incorporations using various artificial AA-tRNAs with all intermediate products being analyzed. Yields within a few seconds displayed similar trends to our prior yields after 30 min without preincubation, demonstrating the relevance of fast kinetics to traditional long-incubation translations. Interestingly, the two anticodon swaps were much less inhibitory in the present optimized system, which should allow more flexibility in the engineering of artificial AA-tRNAs. The biggest kinetic defect was caused by the penultimate dC introduced from the standard, chemoenzymatic, charging method. This prompted peptidyl-tRNA drop-off, decreasing processivities during five consecutive AA incorporations. Indeed, two tRNA charging methods that circumvented the dC dramatically improved efficiencies of ribosomal, consecutive, unnatural AA incorporations to give near wild-type kinetics.
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| 10. |
- Wang, Jinfan, et al.
(författare)
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Kinetics of tRNAPyl-mediated amber suppression in E. coli translation reveals unexpected limiting steps and competing reactions : Kinetics of tRNAPyl-mediated amber suppression
- 2016
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:7, s. 1552-1559
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Tidskriftsartikel (refereegranskat)abstract
- The utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNA(Pyl). Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified Escherichia coli translation system. Dipeptide formation from initiator fMet-tRNA(fMet) and tRNA(Pyl) charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, approximate to 30-fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF-Tu concentration, allowing measurement of K-d values of EF-Tu binding, both of which were approximate to 25-fold weaker than normal. However, binding could be increased approximate to 30-fold by lowering temperature. The fast phase rates were limited by the surprisingly approximate to 10-fold less efficient binding of EF-Tu:GTP:AA-tRNA(Pyl) ternary complex to the ribosomes, not GTP hydrolysis or peptide bond formation. Furthermore, processivity was unexpectedly impaired as approximate to 40% of the dipeptidyl-tRNA(Pyl) could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non-cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. Biotechnol. Bioeng. 2016;113: 1552-1559.
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