在假设的RNA世界中,核酶可以作为现代氨酰基-tRNA合成酶(ARSs)给tRNA充电,从而产生了肽的合成以及原始翻译装置的进化。我们以前报道过一种T-boxzyme,Tx2.1,它在柔性体外翻译(FIT)系统中选择性地将起始tRNA与N-生物素基-苯丙氨酸(BioPhe)原位充电,以产生BioPhe起始肽。这里,我们进行了具有延伸能力的T盒酶(elT盒酶)的体外选择,使用对叠氮基-1-苯丙氨酸(PheAZ)作为酰基供体。我们实施了一种新的策略来富集在3'端选择性自氨基酰化的elT-羧酶-tRNA缀合物。其中一个,elT32可以响应于其同源反密码子以反式将PheAZ充电到tRNA上。elT32的进一步进化导致具有增强的氨基酰化活性的elT49。我们已经证明了含有PheAZ的肽在elT-羧酶整合的FIT系统中的翻译,揭示了elT盒酶能够响应定制翻译系统的同源反密码子原位产生PheAZ-tRNA。这项研究,与Tx2.1一起,说明了一系列核酶可以监督氨基酰化并与原始的基于RNA的翻译系统共同进化的情况。
In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3\'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.