Changes in tertiary structure accompanying a single base change in transfer RNA. Proton magnetic resonance and aminoacylation studies of Escherichia coli tRNAMet f1 and tRNAMet f3 and their spin-labeled (s4U8) derivatives

Abstract

The properties of Escherichia coli tRNAMet f1 and tRNAMet f3 that differ by only one base change, m7G to A at position 47, have been compared structurally by proton magnetic resonance and functionally by the aminoacylation reaction. The NMR spectra of the two tRNA species in the region between 0 and 4 ppm below 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) (methyl and methylene region) were the same except for the absence of the lowest field peak at 3.8 ppm in tRNAMet f3, thus unequivocally identifying this resonance at the methyl group of m7G47 of tRNAMet f1. The same resonance disappears in tRNAMet f1 spin-labeled at s4U8 and reappears in the diamagnetic reduced spin-labeled tRNAMet f1 from which the average distance between the spin-label and the methyl protons of m7G is estimated to be less than 15 A. The proximity of m7G47 but not T55 to s4U8 in the structure of E. coli tRNAMet f1 in solution is consistant with the crystallographic model for yeast tRNAPhe. A spectral comparison of the hydrogen-bond regions (11-14 ppm below DSS) of tRNAMet f1 and tRNAMet f3 reveals major shifts of four resonances previously assigned to tertiary hydrogen bonds. Of the four, the one at lowest field (14.8 ppm) had been assigned by chemical modification to the tertiary (s4U8-A14) hydrogen bond and the one at 13.3 ppm had been tentatively assigned to the tertiary hydrogen bond G23-m7G47 of the 13-23-47 triple. A more positive assignment of the G23-m7G47 at 13.3 ppm could be made from the additional evidence that this resonance, which was first observed in the difference spectrum between spin-labeled tRNAMet f1 and its reduced form, is the only one missing in the analogous difference spectrum of tRNAMet f3. At low ionic strength and in the absence of magnesium ions, the differences in the hydrogen-bonded region of the NMR spectra of tRNAMet f1 and tRNAMet f3 are much greater than in the presence of magnesium ions. The optimal magnesium concentration required for maximal initial velocities is also higher for tRNAMet f3 than for tRNAMet f1. The perturbation caused by the spin-label in destabilizing hydrogen bonds in the region between 13 and 14 ppm is greater for tRNAMet f3 than tRNAMet f1 but the distance relations for the hydrogen bonds in the region between 12 and 13 ppm (the major paramagnetic perturbations) are conserved in the two species. The disruption of one hydrogen bond relative to native tRNAMet f1 either by spin-labeling (s4U8-A14) or by substitution of m7G by A in tRNAMet f3 has little effect on the aminoacyl acceptor activity or the velocity of the aminoacylation reaction at optimal magnesium concentration, but the absence of both tertiary hydrogen bonds in the augmented D-helix region in the spin-labeled tRNAMet f3 results in approximately 60% reduction both in acceptance activity and in initial velocity of the aminoacylation reaction.