Changes in tertiary structure accompanying a single base change in transfer RNA. Proton magnetic resonance and aminoacylation studies of Escherichia coli tRNAMetf1 and tRNAMetf3 and their spin-labeled (S4U8) derivatives

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Abstract
The properties of E. coli tRNAMetf1 and tRNAMetf3 that differ by only 1 base change, m7G to A at position 47, were compared structurally by PMR and functionally by the aminoacylation reaction. The NMR spectra of the 2 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 tRNAMetf3, thus unequivocally identifying this resonance as the methyl group of m7G47 of tRNAMetf1. The same resonance disappears in tRNAMetf1 spin-labeled at s4U[4-thiouridine]8 and reappears in the diamagnetic reduced spin-labeled tRNAMetf1 from which the average distance between the spin-label and the methyl protons of m7G is estimated to be < 15 .ANG.. The proximity of m7G47 but not T55 to s4U8 in the structure of E. coli tRNAMetf1 in solution is consistent with the crystallographic model for yeast tRNAPhe. A spectral comparison of the H-bond regions (11-14 ppm below DSS) of tRNAMetf1 and tRNAMetf3 reveals major shifts of 4 resonances previously assigned to tertiary H bonds. Of the 4, the 1 at lowest field (14.8 ppm) was assigned by chemical modification to the tertiary (s4U8-A14) H bond and the 1 at 13.3 ppm was tentatively assigned to the tertiary H bond G23-m7G47 of the 13-23-47 triple. A more positive assignment of the G23-m7G47 at 13.3 ppm was made from the additional evidence that this resonance, which was first observed in the difference spectrum between spin-labeled tRNAMetf1 and its reduced form, is the only 1 missing in the analogous difference spectrum of tRNAMetf3. At low ionic strength and in the absence of Mg2+, the differences in the H-bonded region of the NMR spectra of tRNAMetf1 and tRMAMetf3 are much greater than in the presence of Mg2+. The optimal Mg2+ concentration required for maximal initial velocities is also higher for tRNAMetf3 than for tRNAMetf1. The perturbation caused by the spin-label in destabilizing H bonds in the region between 13 and 14 ppm is greater for tRNAMetf3 than tRNAMetf1 but the distance relations for the H bonds in the region between 12 and 13 ppm (the major paramagnetic perturbations) are conserved in the 2 spp. The distribution of 1 H bond relative to native tRNAMetf1 either by spin-labling (s4U8-A14) or by substitution of m7G by A in tRNAMetf3 has little effect on the aminoacyl acceptor activity or the velocity of the aminoacylation reaction at optimal Mg2+ concentration, but the absence of both tertiary H bonds in the augmented D-helix region in the spin-labeled tRNAMetf3 results in .apprx. 60% reduction both in acceptance activity and in initial velocity of the aminoacylation reaction.

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