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In Vitro Kinetics of Ribosomal Incorporation of Unnatural Amino Acids
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Forster group)ORCID iD: 0000-0003-4170-9289
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1369
Keyword [en]
ribosome, protein synthesis, unnatural amino acids, kinetics
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-282023ISBN: 978-91-554-9563-3 (print)OAI: oai:DiVA.org:uu-282023DiVA: diva2:919265
Public defence
2016-06-03, BMC A1:111a, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-05-11 Created: 2016-04-01 Last updated: 2016-06-01
List of papers
1. Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro
Open this publication in new window or tab >>Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro
Show others...
2014 (English)In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 9, no 6, 1303-1311 p.Article 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
2. Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements
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, 2187-2192 p.Article 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
3. Kinetics of tRNAPyl-mediated amber suppression in E. coli translation reveals unexpected limiting steps and competing reactions: Kinetics of tRNAPyl-mediated amber suppression
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, 1552-1559 p.Article 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.

Keyword
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

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