Mischarging of M. barkeri tRNA Pyl with alanine and serine in vitro

Abstract

The discovery of pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase specific for the charging of pyrrolysine onto its cognate tRNA species (tRNAPyl) provided the first example of a synthetase evolved for activation of a non-canonical amino acid. The secondary structure of tRNAPyl is unusual for its lengthened anticodon stem and short variable loop. Methanosarcina barkeri has two SerRSs, one of which homologous to bacterial SerRS and a dissimilar one often found in methanogens. In bovine mitochondria, a bacterial type SerRS charges a tRNASer with a very similar secondary structure to tRNAPyl. We tested if the bacterial and methanogenic SerRSs successfully charges the tRNAPyl transcripts with serine, and whether M. barkeri PylRS charges tRNASer with pyrrolysine in vitro and in vivo. Another facet of specificity that was explored concerned the M. barkeri MS tRNAPyl which has the base pair G3-U70, the main identity element of the alanine tRNA:synthetase system. Though this tRNAPyl contains this element, AlaRS did not charge it with alanine. We made multiple mutations in areas of the acceptor stem of tRNAPyl that are also particularly important to AlaRS and we determined if AlaRS charges any of the mutated tRNAPyl.