Functional contacts of a transfer RNA synthetase with 2′-hydroxyl groups in the RNA minor groove

Abstract

THE functional analysis of determinants on RNA has been largely limited to molecules that contain naturally occurring ribonucleotides, so little is known about the role of 2′-hydroxyl groups in protein–RNA recognition. A single base pair (G3·U70) in the acceptor stem of tRNA^Ala is the principal element for specific recognition by Escherichia coli alanine-tRNA synthetase^1,2. This tRNA synthetase aminoacylates small RNA helices that contain the G3·U70 base pair. Furthermore, removal of the G3 exocyclic 2-amino group that projects into the minor groove eliminates aminoacylation³. This 2-amino group is flanked on either side by ribose 2′-hydroxyl groups that line the minor groove. Here we use chemical synthesis to construct 32 helices that make deoxy and O-methyl substitutions of individual and multiple 2′-hydroxyl groups near and beyond the G3·U70 base pair and find that functional 2′-hydroxyl contacts are clustered within a few angstroms of the critical 2-amino group. These contacts are highly specific and make a thermodynamically significant contribution to RNA recognition.