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Publications (10 of 20) Show all publications
Liljeruhm, J., Wang, J., Kwiatkowski, M., Sabari, S. & Forster, A. C. (2019). Kinetics of D-amino acid incorporation in translation. ACS Chemical Biology, 14(2), 204-213
Open this publication in new window or tab >>Kinetics of D-amino acid incorporation in translation
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2019 (English)In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 14, no 2, p. 204-213Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-373508 (URN)10.1021/acschembio.8b00952 (DOI)000459367200009 ()30648860 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-01-15 Created: 2019-01-15 Last updated: 2019-08-01Bibliographically approved
Liljeruhm, J., Funk, S. K., Tietscher, S., Edlund, A. D., Jamal, S., Yuen, P., . . . Forster, A. C. (2018). Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering, 12, Article ID 8.
Open this publication in new window or tab >>Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
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2018 (English)In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed) Published
Abstract [en]

Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2018
Keywords
Chromoprotein, Fluorescent protein, Coral, Escherichia coli, Genetic marker, Reporter gene, Integration, Fitness cost, BioBrick, iGEM
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356454 (URN)10.1186/s13036-018-0100-0 (DOI)000432246200001 ()29760772 (PubMedID)
Funder
VINNOVASwedish Research Council, 349-2006-267Swedish Research Council, 2011-5787Swedish Research Council, 2016-1Swedish Research Council, 2017-04148Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-07-30 Created: 2018-07-30 Last updated: 2019-01-25Bibliographically approved
Wang, J. & Forster, A. C. (2018). Ribosomal incorporation of unnatural amino acids: lessons and improvements from fast kinetics studies. Current opinion in chemical biology, 46, 180-187
Open this publication in new window or tab >>Ribosomal incorporation of unnatural amino acids: lessons and improvements from fast kinetics studies
2018 (English)In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 46, p. 180-187Article, review/survey (Refereed) Published
Abstract [en]

Technologies for genetically programming ribosomal incorporation of unnatural amino acids are expanding and have created many exciting applications. However, these applications are generally limited by low efficiencies of the unnatural incorporations. Here we review our current mechanistic understanding of these limitations delineated from in vitro fast kinetics. Rate limitation occurs by different mechanisms, depending on the classes of the unnatural amino acids and the tRNA adaptors. This new understanding has led to several ways of improving the incorporation efficiencies, as well as challenges of dogma on rate-limiting steps in protein synthesis in natural cells.

Place, publisher, year, edition, pages
Elsevier, 2018
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-369950 (URN)10.1016/j.cbpa.2018.07.009 (DOI)000449131300025 ()30125734 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council, 2016-1Swedish Research Council, 2017-04148
Available from: 2018-12-18 Created: 2018-12-18 Last updated: 2018-12-18Bibliographically approved
Shepherd, T. R., Du, L., Liljeruhm, J., Samudyata, ., Wang, J., Sjödin, M. O. .., . . . Forster, A. C. (2017). De novo design and synthesis of a 30-cistron translation-factor module. Nucleic Acids Research, 45(18), 10895-10905
Open this publication in new window or tab >>De novo design and synthesis of a 30-cistron translation-factor module
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2017 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 18, p. 10895-10905Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2017
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-340137 (URN)10.1093/nar/gkx753 (DOI)000413107400046 ()28977654 (PubMedID)
Funder
NIH (National Institute of Health), R01-AI0727453Swedish Research Council, NT 2011-5787Swedish Research Council, NT 2016-1Swedish Research Council, 2016-1
Available from: 2018-01-29 Created: 2018-01-29 Last updated: 2018-01-29Bibliographically approved
Wang, J. & Forster, A. C. (2017). Translational roles of the C75 2 ' OH in an in vitro tRNA transcript at the ribosomal A, P and E sites. Scientific Reports, 7, Article ID 6709.
Open this publication in new window or tab >>Translational roles of the C75 2 ' OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 6709Article in journal (Refereed) Published
Abstract [en]

Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2'OH -> 2'H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNA(ugc)(Ala) drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2'OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2'H did not affect A-to P-site translocation nor peptidyl donor activity of tRNA(ugc)(Ala). Rather, the peptidyl acceptor activity of the A-site Ala-tRNA(ugc)(Ala) and the translocation of the P-site deacylated tRNA(ugc)(Ala) to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2'OH groups in tRNAs in translation.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-332839 (URN)10.1038/s41598-017-06991-6 (DOI)000406366000067 ()28751745 (PubMedID)
Funder
Swedish Research Council, 2011-5787, 2016-1
Available from: 2017-11-09 Created: 2017-11-09 Last updated: 2018-01-13Bibliographically approved
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
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