RNA determinants for translational editing - Mischarging a minihelix substrate by a tRNA synthetase

The fidelity of protein synthesis requires efficient discrimination of amin o acid substrates by aminoacyl-tRNA synthetases, Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl- tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reactio n, which prevents misactivated valine from being stably joined to tRNA(Ile) . The editing reaction is dependent on the presence of tRNA(Ile), which con tains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine, RNA minihelices comprised of just the acceptor-T Psi C helix of tRNA(Ile) are substrates for specific aminoacylation by IleRS, Th ese substrates lack the aforementioned D-loop nucleotides. Because miniheli ces contain determinants for aminoacylation, we thought that they might als o play a role in editing that has not previously been recognized, Here we s how that, in contrast to tRNA(Ile) minihelix(Ile) is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine, In addition, mutations in minihelix(Ile) that enhance or suppress charging with isoleucine do the same with valine, Thus, minihelix(Ile) contains sign als for charging (by Il-eRS) that are independent of the amino acid and, by itself, minihelix(Ile) provides no determinants for editing. An RNA hairpi n that mimics the D-stem/loop of tRNA(Ile) is also unable to induce the hyd rolysis of misactivated valine, both by itself and in combination with mini helix(Ile). Thus, the native tertiary fold of tRNA(Ile) is required to prom ote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent wit h the idea that, during the development of the genetic code, RNA determinan ts for editing were added after the establishment of an aminoacylation syst em.