Atomic Group “Mutagenesis” Reveals Major Groove Fine Interactions of a tRNA Synthetase with an RNA Helix

Abstract

RNA discrimination by aminoacyl-tRNA synthetases involves both major and minor groove interactions with the acceptor stem domain of tRNA substrates. In the case of class II Escherichia coli alanyl-tRNA synthetase (AlaRS), minor groove atomic groups in and around the unique G3:U70 base pair previously have been shown to be critical for recognition. In this work, we probe the role of the first (1:72) base pair in discrimination by AlaRS by incorporating 26 new base pair combinations at this site. We find that atomic groups in the wild-type G1:C72 base pair do not contribute as significantly to positive recognition by AlaRS as the minor groove elements in and around the G:U base pair. Our results, however, are consistent with the importance of major groove discrimination at this site. In particular, substrates with a major groove carbonyl oxygen presented by either a G or a U at position 72 are very poor alanine acceptors. Comparison of inactive N1:G72 duplexAla variants with active N1:2-aminopurine72 varian...