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Lundius, Ebba G.
Publications (2 of 2) Show all publications
Kipper, K., Lundius, E. G., Ćurić, V., Nikic, I., Wiessler, M., Lemke, E. A. & Elf, J. (2017). Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli. ACS Photonics, 6(2), 233-255
Open this publication in new window or tab >>Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli
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2017 (English)In: ACS Photonics, E-ISSN 2330-4022, Vol. 6, no 2, p. 233-255Article in journal (Refereed) Published
Abstract [en]

Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
Keywords
noncanonical amino acid, tetrazine, recoded E. coli, in vivo fluorescence labeling, OmpC, single particle tracking
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-320513 (URN)10.1021/acssynbio.6b00138 (DOI)000394736400009 ()27775882 (PubMedID)
Funder
EU, European Research CouncilSwedish Research CouncilKnut and Alice Wallenberg FoundationGerman Research Foundation (DFG), SPP1623
Available from: 2017-04-20 Created: 2017-04-20 Last updated: 2018-06-14Bibliographically approved
Sanamrad, A., Persson, F., Lundius, E. G., Fange, D., Gynnå, A. H. & Elf, J. (2014). Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid. Proceedings of the National Academy of Sciences of the United States of America, 111(31), 11413-11418
Open this publication in new window or tab >>Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid
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2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 31, p. 11413-11418Article in journal (Refereed) Published
Abstract [en]

Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

Keywords
nucleoid exclusion, transcription-translation coupling, antibiotics, single-molecule tracking, single-molecule imaging
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-229101 (URN)10.1073/pnas.1411558111 (DOI)000339807200043 ()25056965 (PubMedID)
Available from: 2014-07-30 Created: 2014-07-30 Last updated: 2017-12-05Bibliographically approved
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