Cys-tRNAj as a Second Translation Initiator for Priming Proteins with Cysteine in Bacteria
Résumé
We report the construction of an alternative protein priming system to recode genetic translation in Escherichia coli by designing, through trial and error, a chimeric initiator whose sequence identity points partly to elongator tRNA Cys and partly to initiator tRNA f Met . The elaboration of a selection based on the N-terminal cysteine imperative for the function of glucosamine-6phosphate synthase, an essential enzyme in bacterial cell wall synthesis, was a crucial step to achieve the engineering of this Cys-tRNA j . Iterative improvement of successive versions of Cys-tRNA j was corroborated in vitro by using a biochemical luciferase assay and in vivo by selecting for translation priming of E. coli thymidylate synthase. Condensation assays using specific fluorescent reagent FITC-Gly-cyanobenzothiazole provided biochemical evidence of cysteine coding at the protein priming stage. We showed that translation can be initiated, by N-terminal incorporation of cysteine, at a codon other than UGC by expressing a tRNA j with the corresponding anticodon. The optimized tRNA j is now available to recode the priming of an arbitrary subset of proteins in the bacterial proteome with absolute control of their expression and to evolve the use of xenonucleotides and the emergence of a tXNA j in vivo.
Domaines
Sciences du Vivant [q-bio]
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paupelin-vaucelle-et-al-2025-cys-trnaj-as-a-second-translation-initiator-for-priming-proteins-with-cysteine-in-bacteria.pdf (6.54 Mo)
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