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  • 1. Du, Liping
    et al.
    Villarreal, Seth
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymerase2012In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 109, no 4, p. 1043-1050Article in journal (Refereed)
    Abstract [en]

    Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TF large (class I) terminator with adjacent copies of the vesicular stomatitis virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within Escherichia coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e., more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TF terminators for BioBrick synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.

  • 2.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Synthetic biology challenges long-held hypotheses in translation, codon bias and transcription2012In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 7, no 7, p. 835-845Article, review/survey (Refereed)
    Abstract [en]

    Synthetic biology is a powerful experimental approach, not only for developing new biotechnology applications, but also for testing hypotheses in basic biological science. Here, examples from our research using the best model system, Escherichia coli, are reviewed. New evidence drawn from synthetic biology has overturned several long-standing hypotheses regarding the mechanisms of transcription and translation: (i) all native aminoacyl-tRNAs are not equally efficient in translation at equivalent concentrations; (ii) accommodation is not always rate limiting in translation, and may not be for any aminoacyl-tRNA; (iii) proline is the only N-alkyl-amino acid in the genetic code not because of special suitability for protein structure, but because of its comparatively high nucleophilicity; (iv) the usages of most sense codons in E. coli do not correlate with cognate tRNA abundances and (v) class II transcriptional pausing and termination by T7 RNA polymerase cannot be assumed to occur in vivo based on in vitro data. Implications of these conclusions for the biotechnology field are discussed.

  • 3.
    Forster, Anthony C.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lee, Sang Yup
    Editorial: NextGen SynBio has arrived...2012In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 7, no 7, p. 827-827Article in journal (Other academic)
  • 4.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids2014In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 20, no 5, p. 632-643Article in journal (Refereed)
  • 5.
    Ieong, Ka-Weng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 43, p. 17955-17962Article in journal (Refereed)
    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.

  • 6.
    Kwiatkowski, Marek
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wang, Jinfan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Facile Synthesis of N-Acyl-aminoacyl-pCpA for Preparation of Mischarged Fully Ribo tRNA2014In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 11, p. 2086-2091Article in journal (Refereed)
    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.

  • 7.
    Liljeruhm, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Funk, Saskia K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tietscher, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Edlund, Anders D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Jamal, Sabri
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Yuen, Pikkei
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Dyrhage, Karl
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Gynnå, Arvid H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Ivermark, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Lövgren, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Törnblom, Viktor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundin, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Wistand-Yuen, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology2018In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed)
    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.

  • 8.
    Liljeruhm, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gullberg, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Synthetic Biology: A Lab Manual2014 (ed. 1st)Book (Refereed)
  • 9.
    Punekar, Avinash S
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Liljeruhm, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Shepherd, Tyson R
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Forster, Anthony C
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 20, p. 9537-9548Article in journal (Refereed)
    Abstract [en]

    RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

  • 10.
    Punekar, Avinash S
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Shepherd, Tyson R
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Liljeruhm, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 20, p. 10507-20Article in journal (Refereed)
    Abstract [en]

    RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2'O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM-AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2'O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.

  • 11. Quax, Tessa E. F.
    et al.
    Wolf, Yuri I.
    Koehorst, Jasper J.
    Wurtzel, Omri
    van der Oost, Richard
    Ran, Wenqi
    Blombach, Fabian
    Makarova, Kira S.
    Brouns, Stan J. J.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wagner, Gerhart
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sorek, Rotem
    Koonin, Eugene V.
    van der Oost, John
    Differential Translation Tunes Uneven Production of Operon-Encoded Proteins2013In: Cell Reports, ISSN 2211-1247, Vol. 4, no 5, p. 938-944Article in journal (Refereed)
    Abstract [en]

    Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.

  • 12.
    Shepherd, Tyson R
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. MIT, Dept Biol Engn, Cambridge, MA 02139 USA..
    Du, Liping
    Vanderbilt Univ, Med Ctr, Dept Pharmacol, Nashville, TN 37232 USA.;Vanderbilt Univ, Med Ctr, Ctr Quantitat Sci, Nashville, TN 37232 USA..
    Liljeruhm, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Samudyata,
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wang, Jinfan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Sjödin, Marcus O.D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Swedish Toxicol Sci Res Ctr Swetox, S-15136 Sodertalje, Sweden..
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. GE Healthcare Biosci, Life Sci, S-75184 Uppsala, Sweden..
    Yomo, Tetsuya
    East China Normal Univ, Inst Biol & Informat Sci, Sch Comp Sci & Software Engn, Sch Life Sci, Shanghai 200062, Peoples R China..
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Vanderbilt Univ, Med Ctr, Dept Pharmacol, Nashville, TN 37232 USA..
    De novo design and synthesis of a 30-cistron translation-factor module2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 18, p. 10895-10905Article in journal (Refereed)
    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.

  • 13. Wang, Harris H.
    et al.
    Huang, Po-Yi
    Xu, George
    Haas, Wilhelm
    Marblestone, Adam
    Li, Jun
    Gygi, Steven P.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jewett, Michael C.
    Church, George M.
    Multiplexed in Vivo His-Tagging of Enzyme Pathways for in Vitro Single-Pot Multienzyme Catalysis2012In: ACS Synthetic Biology, ISSN 2161-5063, Vol. 1, no 2, p. 43-52Article in journal (Refereed)
    Abstract [en]

    Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced in vitro reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism Escherichia coli to reconstitute functional synthetic proteomes in vitro. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes in vivo that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation, protein complex enabled protein synthesis in vitro. Our approach can be applied to a growing area of applications in in vitro one-pot multienzyme catalysis (MEC) to manipulate or enhance in vitro pathways such as natural product or carbohydrate biosynthesis.

  • 14.
    Wang, Jinfan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Translational roles of the C75 2 ' OH in an in vitro tRNA transcript at the ribosomal A, P and E sites2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 6709Article in journal (Refereed)
    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.

  • 15.
    Wang, Jinfan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements2015In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 10, no 10, p. 2187-2192Article in journal (Refereed)
    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.

  • 16.
    Wang, Jinfan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kinetics of tRNAPyl-mediated amber suppression in E. coli translation reveals unexpected limiting steps and competing reactions: Kinetics of tRNAPyl-mediated amber suppression2016In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 113, no 7, p. 1552-1559Article in journal (Refereed)
    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.

  • 17.
    Wang, Jinfan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kwiatkowski, Marek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael Yu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Forster, Anthony
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNAPro2014In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 9, no 6, p. 1303-1311Article in journal (Refereed)
  • 18. Watts, R. Edward
    et al.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Update on pure translation display with unnatural amino acid incorporation2012In: Methods in Molecular Biology, ISSN 1064-3745, Vol. 805, p. 349-365Article, review/survey (Refereed)
    Abstract [en]

    The identification of peptide and protein ligands by directed evolution in vitro has been of enormous utility in molecular biology and biotechnology. However, the translation step in almost all polypeptide selection methods is performed in vivo or in crude extracts, restricting applications. These restrictions include a limited library size due to transformation efficiency, unwanted competing reactions in translation, and an inability to incorporate multiple unnatural amino acids (AAs) with high fidelity and efficiency. These restrictions can be addressed by "pure translation display" where the translation step is performed in a purified system. To date, all pure translation display selections have coupled genotype to phenotype in a ribosome display format, though other formats also should be practical. Here, we detail the original, proof-of-principle, pure-translation-display method because this version should be the most suitable for encoding multiple unnatural AAs per peptide product toward the goal of "peptidomimetic evolution." Challenges and progress toward this ultimate goal are discussed and are mainly associated with improving the efficiency of ribosomal polymerization of multiple unnatural AAs.

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