Subtle Functional Interactions in the RNA Minor Groove at a Nonessential Base Pair

We report here a remarkable example of functional interactions with a nonessential base pair in an RNA helix. The results suggest that interactions at a "nonconserved" pair can contribute significantly to the specificity of RNA-protein interactions. Aminoacyl-tRNA synthetases specific for alanine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, methionine, serine, tyrosine, and valine catalyze the sequence-specific aminoacylation of short helical RNAs that mimic the acceptor stem of their cognate tRNAs. In these cases, the helical RNA substrates are composed of as few as four base pairs affixed to the common single-stranded NCCA sub(OH) present at the 3'-end of all tRNAs. The specificity of these reactions remains high even though the efficiency of aminoacylation is reduced relative to that observed with full tRNA. The sequences/structures embedded within tRNA acceptor stems constitute an "operational RNA code" for amino acids. This code may have predated the genetic code. A G3:U70 base pair within the acceptor stem of tRNA super(Ala) is a major determinant for aminoacylation with alanine. The unpaired, exocyclic 2-amino group of G3 marks RNA substrates for alanine acceptance and contributes more than 3 kcal/mol to transition-state stabilization for aminoacylation. (This contribution is significantly greater than that which might be attributed to a helix distortion.) The 2'-OHs at positions 4, 70, and 71 are also important for RNA recognition by alanyl-tRNA synthetase (AlaRS), with each contributing between 1 and 2 kcal/mol to transition-state stabilization. These elements of RNA recognition form a cluster of atoms that is centered around the essential 2-amino group of G3.