RNA tetraloops as minimalist substrates for aminoacylation.
Previous work established that seven-base-pair hairpin microhelices with sequences based on the acceptor stems of alanine, glycine, methionine, and histidine tRNAs can be aminoacylated specifically with their cognate amino acids. To obtain "minimalist" substrates with fewer base pairs, we took advantage of the high thermodynamic stability of RNA tetraloop motifs that are found in ribosomal RNAs. We show here that rationally designed RNA tetraloops with as few as four base pairs are substrates for aminoacylation. Major nucleotide determinants for recognition by the class II synthetases were incorporated into each of the respective tetraloop substrates, resulting in specific aminoacylation by the alanine, glycine, and histidine tRNA synthetases. An analysis of the kinetics of aminoacylation shows that, for the alanine system, the majority of the transition-state stabilization provided by the synthetase-tRNA interaction is reproduced by the interaction of the synthetase with nucleotides in its minimalist tetraloop substrate. In an extension of this work, we also observed specific aminoacylation with the class I methionine tRNA synthetase of RNA tetraloops based on sequences in the acceptor stem of methionine tRNA. Thus, the results demonstrate four different examples where specific aminoacylation is directed by sequences/structures contained in less than half of a turn of an RNA helix.
Shi
J P
JP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Martinis
S A
SA
Schimmel
P
P
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012335:RNA, Ribosomal / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D012335:RNA, Ribosomal; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases