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  • 1.
    Kjellander, Marcus
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Gotz, Kathrin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Liljeruhm, Josefine
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Boman, Mats
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Oorganisk kemi.
    Johansson, Gunnar
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina2013Inngår i: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 35, nr 4, s. 585-590Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Alcohol oxidase from Pichia pastoris was immobilized on nanoporous aluminium oxide membranes by silanization and activation by carbonyldiimidazole to create a flow-through enzyme reactor. Kinetic analysis of the hydrogen peroxide generation was carried out for a number of alcohols using a subsequent reaction with horseradish peroxidase and ABTS. The activity data for the immobilized enzyme showed a general similarity with literature data in solution, and the reactor could generate 80 mmol H2O2/h per litre reactor volume. Horseradish peroxidase was immobilized by the same technique to construct bienzymatic modular reactors. These were used in both single pass mode and circulating mode. Pulsed injections of methanol resulted in a linear relation between response and concentration, allowing quantitative concentration measurement. The immobilized alcohol oxidase retained 58 % of initial activity after 3 weeks of storage and repeated use.

  • 2.
    Liljeruhm, Josefine
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Exotic Ribosomal Enzymology2019Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    This thesis clarifies intriguing enzymology of the ribosome, the multiRNA/multiprotein complex that catalyzes protein synthesis (translation). The large ribosomal RNAs (23S and 16S rRNAs in E. coli) are post-transcriptionally modified by many specific modification enzymes, yet the functions of the modifications remain enigmatic. A deeper insight into two of the 23S rRNA S-adenosyl-methionine-requiring methyltransferase enzymes, RlmM and RlmJ, was given by investigating substrate specificity in vitro. Both enzymes were able to methylate in vitro-transcribed, modification-free, protein-free, 2659-nucleotide-long 23S rRNA. Furthermore, RlmM was able to methylate the 611-nucleotide-long Domain V of the 23S rRNA alone and RlmJ could modify the A2030 with only 25 surrounding nucleotides.

    Translation is evolutionary optimized to incorporate L-amino acids to the exclusion of D-amino acids in the cell. To understand how, and how to engineer around this restriction for pharmacological applications, detailed kinetics of ribosomal dipeptide formation with D- versus L-phenylalanine-tRNA were determined. This was done by varying the concentrations of EF-Tu (which delivers aminoacyl-tRNAs to the ribosome) and the ribosome, as well as changing the tRNA adaptor. Binding to EF-Tu was shown to be rate limiting for D-Phe-tRNA at a low concentration of EF-Tu. Surprisingly, at a higher (physiological) concentration of EF-Tu, binding and subsequent dipeptide synthesis became so efficient that D-Phe incorporation became competitive with L-Phe, and accommodation/peptide bond formation was unmasked as a new rate-limiting step. This highlighted the importance of D-aminoacyl-tRNA deacylase in restricting translation with D-amino acids in vivo.

    Although polypeptides are intrinsically colorless, it is remarkable that evolution has nevertheless enabled ribosomes to synthesize highly colored proteins (chromoproteins). Such eukaryotic proteins reside in coral reefs and undergo self-catalyzed, intramolecular, chromophore formation by reacting with oxygen in a manner highly similar to that of green fluorescent protein. The potential utility of different colored chromoproteins in E. coli was analyzed via codon-optimized over-expression and quantification of maturation times, color intensities and cellular fitness costs. No chromoprotein was found to have the combined characteristics of fast maturation, intense color and low fitness cost. However, semi-rational mutagenesis created different colored variants with identical fitness costs suitable for competition assays and teaching.

    Delarbeid
    1. Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
    Åpne denne publikasjonen i ny fane eller vindu >>Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
    Vise andre…
    2012 (engelsk)Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 20, s. 10507-20Artikkel i tidsskrift (Fagfellevurdert) Published
    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.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-187880 (URN)10.1093/nar/gks727 (DOI)000310970700054 ()22923526 (PubMedID)
    Tilgjengelig fra: 2012-12-11 Laget: 2012-12-11 Sist oppdatert: 2019-01-25bibliografisk kontrollert
    2. Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
    Åpne denne publikasjonen i ny fane eller vindu >>Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
    Vise andre…
    2013 (engelsk)Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, nr 20, s. 9537-9548Artikkel i tidsskrift (Fagfellevurdert) Published
    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.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-211566 (URN)10.1093/nar/gkt719 (DOI)000326746400036 ()23945937 (PubMedID)
    Tilgjengelig fra: 2013-11-26 Laget: 2013-11-26 Sist oppdatert: 2019-01-25bibliografisk kontrollert
    3. Kinetics of D-amino acid incorporation in translation
    Åpne denne publikasjonen i ny fane eller vindu >>Kinetics of D-amino acid incorporation in translation
    Vise andre…
    2019 (engelsk)Inngår i: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 14, nr 2, s. 204-213Artikkel i tidsskrift (Fagfellevurdert) 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.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-373508 (URN)10.1021/acschembio.8b00952 (DOI)000459367200009 ()30648860 (PubMedID)
    Forskningsfinansiär
    Swedish Research Council
    Tilgjengelig fra: 2019-01-15 Laget: 2019-01-15 Sist oppdatert: 2019-08-01bibliografisk kontrollert
    4. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
    Åpne denne publikasjonen i ny fane eller vindu >>Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
    Vise andre…
    2018 (engelsk)Inngår i: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, artikkel-id 8Artikkel i tidsskrift (Fagfellevurdert) 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.

    sted, utgiver, år, opplag, sider
    BIOMED CENTRAL LTD, 2018
    Emneord
    Chromoprotein, Fluorescent protein, Coral, Escherichia coli, Genetic marker, Reporter gene, Integration, Fitness cost, BioBrick, iGEM
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-356454 (URN)10.1186/s13036-018-0100-0 (DOI)000432246200001 ()29760772 (PubMedID)
    Forskningsfinansiär
    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
    Tilgjengelig fra: 2018-07-30 Laget: 2018-07-30 Sist oppdatert: 2019-01-25bibliografisk kontrollert
  • 3.
    Liljeruhm, Josefine
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Funk, Saskia K.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Tietscher, Sandra
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Edlund, Anders D.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. 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 universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär systembiologi. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Ivermark, Katarina
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Lövgren, Jessica
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Törnblom, Viktor
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Virtanen, Anders
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundin, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Wistand-Yuen, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Forster, Anthony C.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology2018Inngår i: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, artikkel-id 8Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 4.
    Liljeruhm, Josefine
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Wang, Jinfan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Kwiatkowski, Marek
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Sabari, Samudra
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Forster, Anthony C.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Kinetics of D-amino acid incorporation in translation2019Inngår i: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 14, nr 2, s. 204-213Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 5.
    Punekar, Avinash S
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Liljeruhm, Josefine
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Shepherd, Tyson R
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Forster, Anthony C
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ2013Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, nr 20, s. 9537-9548Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 6.
    Punekar, Avinash S
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Shepherd, Tyson R
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Liljeruhm, Josefine
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Forster, Anthony C
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA2012Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 20, s. 10507-20Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 7.
    Shepherd, Tyson R
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. 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 universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Samudyata,
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Wang, Jinfan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Sjödin, Marcus O.D.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Swedish Toxicol Sci Res Ctr Swetox, S-15136 Sodertalje, Sweden..
    Wetterhall, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. 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 universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Vanderbilt Univ, Med Ctr, Dept Pharmacol, Nashville, TN 37232 USA..
    De novo design and synthesis of a 30-cistron translation-factor module2017Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 18, s. 10895-10905Artikkel i tidsskrift (Fagfellevurdert)
    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.

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