NMR analysis of bovine tRNA(Trp).

摘要

Nuclear Magnetic Resonance (NMR) was used to study the solution structure of bovine tRNATrp hyperexpressed in Escherichia coli . With the use of 15N-labelling and site-directed mutagenesis to assign overlapping resonances through the basepair replacement of U71A2 by G2C71, U27A43 by G27C43 and G12C23 by U12A23, 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 NOESY and HSQC methods. When the discriminator base A73 and the G12C23 basepair on the D stem, two identity elements on bovine tRNATrp, which are important for effective recognition by TrpRS, 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. Thus A73 appears to be directly recognized by TrpRS, and G12C23 represents an important structural determinant.;Mg2+ effects on the assigned resonances of imino protons allowed the identification of strong, medium and weak Mg2+ binding sites in tRNATrp. Strong Mg2+ binding modes were associated with the residues G7, s4U8, G12 and U52. The observations that G42 was associated with strong Mg2+ binding in only the U12A23-mutant tRNATrp but not the wildtype or G73-mutant tRNATrp, and that the G7, s4U8, G24 and G22 imino protons are associated with a two-site Mg2+ binding mode in wildtype and G73-mutant but only a one-site mode in the U12A23-mutant established the occurrence of conformational change in the U12A23-mutant tRNATrp . They also establish the dependence of Mg2+ binding on tRNA conformation, and the usefulness of Mg2+ binding sites as conformational probes. The thermal titration of tRNATrp in the presence and absence of 10 mM Mg2+ indicated that overall tRNATrp structure stability was increased by more than 15°C by the presence of Mg2+.;NMR method was also used to reveal the recognition mechanism between tRNATrp and TrpRS by comparing the 15N- 1H HSQC spectra of B. subtilius tRNATrp in the absence and presence of B. stearothermophilus TrpRS. Preliminary results about tRNA comformation changes and recognition sites of TrpRS on tRNATrp were described and analyzed.