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 accepters. Comparison of inactive N1:G72 duplex(Ala) variants with active N1:2-aminopurine72 variants shows that deletion of the 6-keto oxygen and the N1-hydrogen of G72 results in a transition state stabilization of at least 3.0 kcal/mol. This work provides an example of a system that combines minor groove interactions at an internal position with the high selectivity of major groove interactions that are possible at the end of an RNA helix.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry