Importance of discriminator base stacking interactions: molecular dynamicsanalysis of A73 microhelix(Ala) variants

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA(Ala) has b een shown, by analysis of variant minihelix aminoacylation activities, to i nvolve a transition state sensitive to changes in the 'discriminator' base at position 73, Solution NMR has indicated that this single-stranded nucleo tide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the aden ine at position 73 substituted by its non-polar isostere 4-methylindole (M) , Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dyn amics (MD) simulations show that the M substitution improves position 73 ba se stacking over G1, as measured by a stacking lifetime analysis. Additiona l MD simulations of wild-type microhelix(Ala) and six variants reveal a pos itive correlation between N73 base stacking propensity over G1 and aminoacy lation activity. For the two Delta N7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that cont ain N7 and we conclude that the decrease in aminoacylation efficiency obser ved upon deletion of N7 is likely due to loss of a direct stabilizing inter action with the synthetase.