G:U-Independent RNA Minihelix Aminoacylation by Nanoarchaeum equitans Alanyl-tRNA Synthetase: An Insight into the Evolution of Aminoacyl-tRNA Synthetases

Nanoarchaeum equitans is a species of hyperthermophilic archaea with the smallest genome size. Its alanyl-tRNA synthetase genes are split into AlaRS-α and AlaRS-β, encoding the respective subunits. In the current report, we surveyed N. equitans AlaRS-dependent alanylation of RNA minihelices, composed only of the acceptor stem and the T-arm of tRNAAla. Combination of AlaRS-α and AlaRS-β showed a strong alanylation activity specific to a single G3:U70 base pair, known to mark a specific tRNA for charging with alanine. However, AlaRS-α alone had a weak but appreciable alanylation activity that was independent of the G3:U70 base pair. The shorter 16-mer RNA tetraloop substrate mimicking only the first four base pairs of the acceptor stem of tRNAAla, but with C3:G70 base pair, was also successfully aminoacylated by AlaRS-α. The end of the acceptor stem, including CCA-3' terminus and the discriminator A73, was able to function as a minimal structure for the recognition by the enzyme. Our findings imply that aminoacylation by N. equitans AlaRS-α may represent a vestige of a primitive aminoacylation system, before the appearance of the G3:U70 pair as an identity element for alanine.