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Kwiatkowski, Marek
Publications (7 of 7) Show all publications
Wang, J., Kwiatkowski, M. & Forster, A. (2016). Kinetics of tRNAPyl-mediated amber suppression in E. coli translation reveals unexpected limiting steps and competing reactions: Kinetics of tRNAPyl-mediated amber suppression. Biotechnology and Bioengineering, 113(7), 1552-1559
Open this publication in new window or tab >>Kinetics of tRNAPyl-mediated amber suppression in E. coli translation reveals unexpected limiting steps and competing reactions: Kinetics of tRNAPyl-mediated amber suppression
2016 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 113, no 7, p. 1552-1559Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
protein synthesis; tRNA(Pyl); amber suppression; kinetics; EF-Tu; release factor 2
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-270305 (URN)10.1002/bit.25917 (DOI)000377527900017 ()26705134 (PubMedID)
Funder
Swedish Research Council
Available from: 2015-12-26 Created: 2015-12-26 Last updated: 2017-12-01Bibliographically approved
Wang, J., Kwiatkowski, M. & Forster, A. (2015). Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements [Letter to the editor]. ACS Chemical Biology, 10(10), 2187-2192
Open this publication in new window or tab >>Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements
2015 (English)In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 10, no 10, p. 2187-2192Article in journal, Letter (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-262591 (URN)10.1021/acschembio.5b00335 (DOI)000363225100002 ()26191973 (PubMedID)
Funder
Swedish Research CouncilSwedish Research Council
Available from: 2015-09-16 Created: 2015-09-16 Last updated: 2017-12-04
Ieong, K.-W., Pavlov, M. Y., Kwiatkowski, M., Ehrenberg, M. & Forster, A. C. (2014). A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids. RNA: A publication of the RNA Society, 20(5), 632-643
Open this publication in new window or tab >>A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids
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2014 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 20, no 5, p. 632-643Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-218734 (URN)10.1261/rna.042234.113 (DOI)000334677800005 ()
Available from: 2014-02-16 Created: 2014-02-16 Last updated: 2017-12-06Bibliographically approved
Kwiatkowski, M., Wang, J. & Forster, A. C. (2014). Facile Synthesis of N-Acyl-aminoacyl-pCpA for Preparation of Mischarged Fully Ribo tRNA. Bioconjugate chemistry, 25(11), 2086-2091
Open this publication in new window or tab >>Facile Synthesis of N-Acyl-aminoacyl-pCpA for Preparation of Mischarged Fully Ribo tRNA
2014 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 11, p. 2086-2091Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-239993 (URN)10.1021/bc500441b (DOI)000345309000020 ()25338217 (PubMedID)
Available from: 2015-01-05 Created: 2015-01-05 Last updated: 2017-12-05
Wang, J., Kwiatkowski, M., Pavlov, M. Y., Ehrenberg, M. & Forster, A. (2014). Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro. ACS Chemical Biology, 9(6), 1303-1311
Open this publication in new window or tab >>Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro
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2014 (English)In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 9, no 6, p. 1303-1311Article in journal (Refereed) Published
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-229021 (URN)10.1021/cb500036a (DOI)000337870500012 ()
Available from: 2014-07-25 Created: 2014-07-25 Last updated: 2017-12-05
Wu, S., Chen, Y., Mao, G., Trobro, S., Kwiatkowski, M. & Kirsebom, L. A. (2014). Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates. Nucleic Acids Research, 42(1), 631-642
Open this publication in new window or tab >>Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates
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2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 1, p. 631-642Article in journal (Refereed) Published
Abstract [en]

We have used model substrates carrying modified nucleotides at the site immediately 5' of the canonical RNase P cleavage site, the -1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at -1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at -1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at -1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the -1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-221063 (URN)10.1093/nar/gkt853 (DOI)000331136000056 ()
Available from: 2014-03-26 Created: 2014-03-25 Last updated: 2017-12-05Bibliographically approved
Ieong, K.-W., Pavlov, M. Y., Kwiatkowski, M., Forster, A. C. & Ehrenberg, M. (2012). Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation. Journal of the American Chemical Society, 134(43), 17955-17962
Open this publication in new window or tab >>Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation
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2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 43, p. 17955-17962Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-186030 (URN)10.1021/ja3063524 (DOI)000310483500024 ()
Available from: 2012-11-28 Created: 2012-11-27 Last updated: 2017-12-07Bibliographically approved
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