Origins of translation machinery: Aminoacylation of minimalist tRNA(leu) molecules and non-ribosomal peptide bond formation

A series of RNA analogs of Escherichia coli tRNA leu with specific domain deletions and/or point mutations were synthesized. These RNAs were used to study the recognition requirements for aminoacylation by leucyl-tRNA synthetase (LeuRS). The anticodon stem-loop and variable arm domains were both found to be separately dispensable for Ieucylation. Simultaneous deletion of both domains further reduced aminoacylation. It was possible to partially restore activity by using alternative sequences in the nucleotide linker region between the remaining D-stem and T-stem. Point mutations in the D- and T-loops in one of the analogs completely abolished leucylation. Changing A73 to C73 in this minimalist variant also abolished leucylation, confirming that the discriminator base was a major identity element. These results suggest that tertiary interactions in the core region of tRNA leu are essential for recognition by LeuRS. Hydrolytic editing assays with a LeuRS mutant enzyme and each of the aminoacylatable tRNAleu analogs suggest that the variable loop interacts with the enzyme's editing site.;Several avenues by which aminoacylated RNAs might participate in peptide bond formation in the absence of ribosomal components were also investigated. The potential for 5'-leucyl-adenylate anhydrides to form dipeptides was tested. Leu-Leu dipeptides and leucine polypeptides were detected by thin layer chromatography. A published claim that dipeptides could be formed in the presence of aminoacylated tRNA, a corresponding mRNA template, Mg 2+, and Ala-His dipeptide acting as a catalyst was examined in detail. The reactions published were duplicated using [14C]-leucine-tRNA leu and poly-AUUU mRNA, as well as alternative catalysts or substrates. Leu-Leu dipeptide synthesis did not occur in the presence of Ala-His, although two unidentified by-products were formed during the reaction. Unknown product 1 was shown to be a leucyl-ethyl-ester formed by ethanolysis. Unknown product 2 was identified as adenosine-leucine. These results strongly suggest that the published claims may be artifactual.