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Inefficient deliver...
Inefficient delivery but fast peptide bond formation of unnatural l -aminoacyl-tRNAs in translation
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- Ieong, Ka-Weng (författare)
- Uppsala universitet,Struktur- och molekylärbiologi
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- Pavlov, Michael Y. (författare)
- Uppsala universitet,Struktur- och molekylärbiologi
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- Kwiatkowski, Marek (författare)
- Uppsala universitet,Struktur- och molekylärbiologi
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- Forster, Anthony C. (författare)
- Uppsala universitet,Struktur- och molekylärbiologi
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- Ehrenberg, Måns (författare)
- Uppsala universitet,Struktur- och molekylärbiologi
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(creator_code:org_t)
- 2012-10-22
- 2012
- Engelska.
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 134:43, s. 17955-17962
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
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- art (ämneskategori)
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