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Shepherd, Tyson R
Publications (3 of 3) Show all publications
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
Punekar, A. S., Liljeruhm, J., Shepherd, T. R., Forster, A. C. & Selmer, M. (2013). Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ. Nucleic Acids Research, 41(20), 9537-9548
Open this publication in new window or tab >>Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
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2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 20, p. 9537-9548Article in journal (Refereed) 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.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-211566 (URN)10.1093/nar/gkt719 (DOI)000326746400036 ()23945937 (PubMedID)
Available from: 2013-11-26 Created: 2013-11-26 Last updated: 2019-01-25Bibliographically approved
Punekar, A. S., Shepherd, T. R., Liljeruhm, J., Forster, A. C. & Selmer, M. (2012). Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA. Nucleic Acids Research, 40(20), 10507-20
Open this publication in new window or tab >>Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 20, p. 10507-20Article in journal (Refereed) 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.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-187880 (URN)10.1093/nar/gks727 (DOI)000310970700054 ()22923526 (PubMedID)
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2019-01-25Bibliographically approved
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