Structural variability of tRNA: small-angle x-ray scattering of the yeast tRNAphe-Escherichia coli tRNAGlu2 complex.

Abstract

The structure of the complex formed in solution between yeast tRNAPhe and Escherichia coli tRNAGlu has been studied by small-angle x-ray scattering. The complex has a radius of gyration of 4.0 nm and an electron-pair distance distribution that is incompatible with a model composed to two tRNAs joined at their complementary anticodons and exhibiting the L shape seen in the crystal. Instead a model in which the two tRNAs, still bound via the anticodons, assume a conformation with the acceptor arms folded toward the anticodon arms agrees with the observed scattering curves.The crystal structure of tRNAPhe (1, 2) explains most ofthe conserved features of the tRNA nucleotide sequence (3). The emerging picture is, however, a static one and refers to a conformation that may be subject to constraints imposed by the crystal lattice. It has been proposed that the tRNA assumes different conformations during its interactions with other components of the protein synthesis system (4, 5). This hypothesis is supported by nuclease digestion experiments (6, 7), measurements of tRNA diffusion (8, 9), nucleotide binding experiments (10,11), and chemical modification experiments (12) indicating the existence of more than one tRNA conformation, depending on solvent conditions.In particular, interactions between the anticodon and a cognate codon appear to induce alterations ofthe structure (10-12), so that the T loop becomes available for intermolecular interactions. Since, in the crystal structure, the T-P-C-G sequence interacts with the D loop, changes of the tRNA structure have been inferred to account for this observation.We have therefore undertaken experiments using small-angle x-ray scattering to obtain independent information on the solution structure ofcoded tRNA. Previous studies (13-15) have shown that this technique allows the determination ofimportant structural parameters oftRNA in solution. In the present study, we have used tRNAPhe from yeast and tRNA2 lu from Escherichia coli, which form a very stable complex via their complementary anticodons (16)(17)(18). This complex can be considered an instructive model for the study ofeffects ofcodon-anticodon interactions on the solution structure of tRNA.We find that the scattering properties of the tRNAlPhe_ tRNAGlU complex are best explained by a model in which the two tRNAs are joined at their anticodons and the acceptor arms are folded toward the anticodon arms instead of being at right angles to the anticodon arms as they are in the L-shaped crystal structure. MATERIALS AND METHODStRNAPhe from yeast (lots 1309136 and 1209336) and tRNAGlu (lot 1448407) from E. coli were obtained from Boehringer Mannheim. The amino acid acceptancy was-1,280 pmol/A2 unit for tRNA~he and, as specified by the manufacturer, 1,372 pmol/A2w unit for tRNA2 .The tRNAs were dialyzed at 4°C against two changes of each of the following buffers: 10 mM Tris-HCl/0. 1 M KCl/0. 1 mM EDTA, pH 7.5, and 10 mM Tris-HCl/0. 1 M KCl/0. 1 mM EDTA/5 mM MgCl2, pH 7.5. The last dialysis buffer was used for a...

Anticodon-anticodon interaction induces conformational changes in tRNA: yeast tRNAAsp, a model for tRNA-mRNA recognition.

“…The observed conformational change, although observable and significant, is not as large as those postulated by various authors (e.g., see refs. [29][30][31]. Our results suggest a mechanism based on a fine tuning of both anticodons with a transfer of flexibility to the end of the arm in the D-and T-loop region.…”

Determining RNA three-dimensional structures using low-resolution data

“…the lengths of the probes are comparable to its radius of gyration. Given that during tRNA folding many conformations can be explored [45], it would be interesting to compare buffer solutions between the MPE-based probing and that of SAXS, for instance, or to revisit how the EDTA-based probing data should be interpreted. That being said, the radius of gyration of the tRNA has been measured to be about 23.5 Å [46], which is within the range of the tether probe.…”

Thermodynamic Data for Biochemistry and Biotechnology

“…If again the main part of this slope can be explained by the residual structure of poly(C), this coincidence need not be accidental, since poly(C) has almost the same enthalpy of unstacking as poly(A) and its residual structure is nearly 10K more stable. This observation seems even more striking in the light of a subsequent publication of the same authors in which they report the remarkable dependence on temperature of the heat of the reaction between poly(BrC) and poly(l) at almost the same salt conditions [56], although it is known that poly(BrC) has a more stable structure and therefore is more stacked in the (298)(299)(300)(301)(302)(303)(304)(305)(306)(307)(308)(309)(310) K temperature range [32]. This observation seems even more striking in the light of a subsequent publication of the same authors in which they report the remarkable dependence on temperature of the heat of the reaction between poly(BrC) and poly(l) at almost the same salt conditions [56], although it is known that poly(BrC) has a more stable structure and therefore is more stacked in the (298)(299)(300)(301)(302)(303)(304)(305)(306)(307)(308)(309)(310) K temperature range [32].…”