uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Kinetics of D-amino acid incorporation in translation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.ORCID iD: 0000-0003-4170-9289
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2019. Vol. 14, no 2, p. 204-213
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-373508DOI: 10.1021/acschembio.8b00952ISI: 000459367200009PubMedID: 30648860OAI: oai:DiVA.org:uu-373508DiVA, id: diva2:1278764
Funder
Swedish Research CouncilAvailable from: 2019-01-15 Created: 2019-01-15 Last updated: 2019-03-21Bibliographically approved
In thesis
1. Exotic Ribosomal Enzymology
Open this publication in new window or tab >>Exotic Ribosomal Enzymology
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 43
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1770
Keywords
rRNA modification, Methyltransferase, RlmM, RlmJ, D-amino acid, Unnatural amino acid, Chromoprotein
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-374965 (URN)978-91-513-0567-7 (ISBN)
Public defence
2019-03-08, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-02-15 Created: 2019-01-25 Last updated: 2019-03-18

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMed

Authority records BETA

Liljeruhm, JosefineWang, JinfanKwiatkowski, MarekForster, Anthony C.

Search in DiVA

By author/editor
Liljeruhm, JosefineWang, JinfanKwiatkowski, MarekForster, Anthony C.
By organisation
Molecular Biology
In the same journal
ACS Chemical Biology
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 63 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf