Paupelin-Vaucelle Humbeline, Boschiero Claire, Lazennec-Schurdevin Christine, Schmitt Emmanuelle, Mechulam Yves, Marlière Philippe, Pezo Valérie
Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 2 rue Gaston Crémieux, 91057 Evry, France.
Laboratoire de Biologie Structurale de la Cellule, BIOC, Ecole polytechnique, CNRS, Institut Polytechnique de Paris, Bat 84, Route de Saclay, 91128 Palaiseau cedex, France.
ACS Omega. 2025 Jan 29;10(5):4548-4560. doi: 10.1021/acsomega.4c08326. eCollection 2025 Feb 11.
We report the construction of an alternative protein priming system to recode genetic translation in by designing, through trial and error, a chimeric initiator whose sequence identity points partly to elongator tRNA and partly to initiator tRNA . The elaboration of a selection based on the N-terminal cysteine imperative for the function of glucosamine-6-phosphate synthase, an essential enzyme in bacterial cell wall synthesis, was a crucial step to achieve the engineering of this Cys-tRNA. Iterative improvement of successive versions of Cys-tRNA was corroborated by using a biochemical luciferase assay and by selecting for translation priming of 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 with the corresponding anticodon. The optimized tRNA 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 .
我们报告了一种替代性蛋白质起始系统的构建，该系统通过反复试验设计了一种嵌合起始子，其序列部分与延伸因子tRNA相同，部分与起始因子tRNA相同，从而对基因翻译进行重新编码。基于氨基葡萄糖-6-磷酸合酶（细菌细胞壁合成中的一种必需酶）功能所需的N端半胱氨酸构建筛选方法，是实现这种半胱氨酸tRNA工程的关键一步。通过使用生化荧光素酶测定法以及选择胸苷酸合成酶的翻译起始，证实了连续版本的半胱氨酸tRNA的迭代改进。使用特异性荧光试剂FITC-甘氨酸-氰基苯并噻唑进行的缩合测定提供了蛋白质起始阶段半胱氨酸编码的生化证据。我们表明，通过表达具有相应反密码子的tRNA，通过N端掺入半胱氨酸，可以在UGC以外的密码子处起始翻译。现在，经过优化的tRNA可用于对细菌蛋白质组中任意蛋白质子集的起始进行重新编码，从而绝对控制其表达，并用于开发异源核苷酸的使用以及tXNA的出现。
