| 1. |
- Shepherd, Tyson R, et al.
(författare)
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De novo design and synthesis of a 30-cistron translation-factor module
- 2017
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 45:18, s. 10895-10905
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 2. |
- Wang, Jinfan, et al.
(författare)
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Translational roles of the C75 2 ' OH in an in vitro tRNA transcript at the ribosomal A, P and E sites
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2'OH -> 2'H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNA(ugc)(Ala) drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2'OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2'H did not affect A-to P-site translocation nor peptidyl donor activity of tRNA(ugc)(Ala). Rather, the peptidyl acceptor activity of the A-site Ala-tRNA(ugc)(Ala) and the translocation of the P-site deacylated tRNA(ugc)(Ala) to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2'OH groups in tRNAs in translation.
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