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Mischarging of M. barkeri tRNA Pyl with alanine and serine in vitro
Author(s) -
Li Darrick,
Polycarpo Carla,
Ambrogelly Alexandre,
Söll Dieter
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a503-c
Subject(s) - methanosarcina barkeri , alanine , serine , transfer rna , amino acid , biochemistry , biology , aminoacylation , threonine , chemistry , enzyme , rna , genetics , gene , bacteria , methanogenesis
The discovery of pyrrolysyl‐tRNA synthetase (PylRS), an aminoacyl‐tRNA synthetase specific for the charging of pyrrolysine onto its cognate tRNA species (tRNA Pyl ) provided the first example of a synthetase evolved for activation of a non‐canonical amino acid. The secondary structure of tRNA Pyl 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 tRNA Ser with a very similar secondary structure to tRNA Pyl . We tested if the bacterial and methanogenic SerRSs successfully charges the tRNA Pyl transcripts with serine, and whether M. barkeri PylRS charges tRNA Ser with pyrrolysine in vitro and in vivo . Another facet of specificity that was explored concerned the M. barkeri MS tRNA Pyl which has the base pair G3‐U70, the main identity element of the alanine tRNA:synthetase system. Though this tRNA Pyl contains this element, AlaRS did not charge it with alanine. We made multiple mutations in areas of the acceptor stem of tRNA Pyl that are also particularly important to AlaRS and we determined if AlaRS charges any of the mutated tRNA Pyl .