Overlapping Nucleotide Determinants for Specific Aminoacylation of RNA Microhelices

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Abstract
A seven-base pair microhelix that recapitulates a glycine transfer RNA (tRNA) acceptor helix can be specifically aminoacylated with glycine. A single base pair and the single-stranded discriminator base near the attachment site are essential for aminoacylation. These nucleotide sequence elements, and those in microhelices that can be charged with histidine and alanine, occur in the same positions and therefore overlap. Studies on a systematic set of sequence variants showed that no microhelix could be charged with more than one amino acid. Also, none of the three cognate aminoacyl-tRNA synthetases (aaRSs) gave a detectable amount of aminoacylation of the CCA trinucleotide that is common to the 3' ends of all tRNAs, showing that the specific acceptor stem nucleotide bases confer aminoacylation. An analysis of the relative contributions of these microhelices to overall tRNA recognition indicated that their interaction with aaRSs constitutes a substantial part of the recognition of the whole tRNAs.