Activation of microhelix charging by localized helix destabilization

Abstract

We report that aminoacylation of minimal RNA helical substrates is enhanced by mismatched or unpaired nucleotides at the first position in the helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem of initiator and elongator tRNAs with greatly reduced efficiency relative to full-length tRNA substrates. The cocrystal structure of the class I glutaminyl-tRNA synthetase with tRNAGln revealed an uncoupling of the first (1.72) base pair of tRNAGln, and tRNAMet was proposed by others to have a similar base-pair uncoupling when bound to methionyl-tRNA synthetase. Because the anticodon is important for efficient charging of methionine tRNA, we thought that 1.72 distortion is probably effected by the synthetase-anticodon interaction. Small RNA substrates (minihelices, microhelices, and duplexes) are devoid of the anticodon triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test this hypothesis, we constructed microhelices that vary in their ability to form a 1.72 base pair. The results of kinetic assays show that microhelix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is also seen with the analogous deletion in a minihelix substrate for the closely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in part for the lack of presumptive anticodon-induced acceptor stem distortion.

Figure 1

Substrates for E. coli MetRS. (Top) Sequences of E. coli elongator and initiator tRNAs. The base pairs at the first position of the acceptor stems are shaded. (Middle) Microhelix substrates that were assayed. Wild-type microhelix^fMet and microhelix^Met substrates and variants of microhelix^Met are shown, with substitutions of the first base pair being shaded. (Bottom) Duplex^Met substrates, with base substitutions shaded.

Figure 2

Aminoacylation of microhelix and duplex substrates of MetRS. The substitution of the A73 discriminator base abolishes aminoacylation.

Figure 3

Enhancement of microhelix^Met aminoacylation by 1⋅72 base-pair mismatches.

Figure 4

Enhancement of aminoacylation by terminal nucleotide deletion. (Upper) Acid gel analysis of microhelix aminoacylation showing the enhanced aminoacylation of the Δ1⋅C72 microhelix^Met substrate with [³⁵S]methionine after a 15-min reaction. (Lower) Time course of aminoacylation of microhelix^Met and Δ1⋅C72 microhelix^Met. The enhanced aminoacylation conferred by the Δ1⋅C72 substitution is also seen with the duplex^Met substrate (Inset).

Figure 5

Enhancement of minihelix^Ile aminoacylation by deletion of nucleotide A1. (Upper) Minihelix^Ile recapitulates the acceptor stem and TΨC loop of tRNA^Ile. Δ1⋅U72 minihelix^Ile is lacking the 5′ terminal adenosine. (Lower) Time course of aminoacylation of minhelix^Ile and Δ1⋅U72 minihelix^Ile.