Tertiary core rearrangements in a tight binding transfer RNA aptamer

Guided by an in vitro selection experiment designed to obtain tight binding aptamers of Escherichia coli glutamine specific tRNA (tRNAGln) for glutaminyl-tRNA synthetase (GlnRS), we have engineered a tRNA mutant in which the five-nucleotide variable loop sequence 5′-44CAUUC48-3′ is replaced by 5′-44AGGU48-3′. This mutant tRNA binds to GlnRS with 30-fold improved affinity compared to the wild type. The 2.7 Å cocrystal structure of the RNA aptamer–GlnRS complex reveals major rearrangements in the central tertiary core of the tRNA, while maintaining an RNA–protein interface identical to the wild type. The repacked RNA core features a novel hydrogen bonding arrangement of the trans Levitt pair G15–U48, a new sulfate binding pocket in the major groove, and increased hydrophobic stacking interactions among the bases. These data suggest that enhanced protein binding to a mutant globular RNA can arise from stabilization of RNA tertiary interactions rather than optimization of RNA–protein contacts.

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