Abstract
NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).
Highlights
NMR was used to study the solution structure of bovine tRNATrp hyperexpressed in Escherichia coli
When the discriminator base A73 and the G12C23 base pair on the D stem, two identity elements on bovine tRNATrp that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G73 and U12A23, respectively, NMR analysis revealed an important conformational change in the U12A23 mutant but not in the G73 mutant molecule
The observations that G42 was associated with strong Mg2؉ binding in only the U12A23 mutant tRNATrp but not the wild type or G73 mutant tRNATrp and that the G7, s4U8, G24, and G22 imino protons are associated with a two-site Mg2؉ binding mode in wild type and G73 mutant but only a one-site mode in the U12A23 mutant established the occurrence of conformational change in the U12A23 mutant tRNATrp
Summary
NMR was used to study the solution structure of bovine tRNATrp hyperexpressed in Escherichia coli. By mutagenizing a base pair with a resonance that overlaps with that of another base pair, the latter resonance may be analyzed unambiguously Use of this approach has made possible the assignment of almost all of the imino protons in the helical regions and the tertiary base pairs in Bacillus subtilis tRNATrp. Magnesium ions are essential to tRNA function, and their binding to tRNA has long been investigated [16]. Given the extensive imino proton assignments made possible by a combination of tRNA sequence mutagenesis and 2D1 NMR [15], the possible conformational roles of the A73 and G12C23 identity elements on bovine tRNATrp were examined in the present study based on their mutation to the ineffective forms of G73 and U12A23, respectively, and monitoring NMR chemical shift changes of different imino protons in the wild type and mutant molecules. Conformational changes were detected through changes in the behavior of strong Mg2ϩ binding sites
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