Naturally-occurring modification restricts the anticodon domain conformational space of tRNAPhe

Post-transcriptional modifications contribute chemistry and structure to RNAs. Modifications of tRNA at nucleoside 37, 3'-adjacent to the anticodon, are particularly interesting because they facilitate codon recognition and negate translational frame-shifting. To assess if the functional contribution of a position 37-modified nucleoside defines a specific structure or restricts conformational flexibility) structures of the yeast tRNA (Phe) anticodon stem and loop (ASL(Phe)) with naturally occurring modified nucleosides differing only at position 37, ASL(Phe)-(Cm-32,Gm(34),m(5)C(40)), and ASL(Phe)-(CM31,Gm(34),m(1)G(37),m(5)C(40)). were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD similar to 0.6 Angstrom) of five canonical base-pairs in standard A-form RNA. The NOE walk was evident on the 5' and 3' sides of the stems of both RNAs, and extended to the adjacent loop nucleosides. The NOESY cross-peaks involving U-33, H2' and characteristic of tRNA`s anticodon domain U-turn were present but weak, whereas those involving the U-33 H1' proton were absent from the spectra of both ASLs. However, ASL(Phe)-(Cm-32,Gm(34),m(1)G(37), m(5)C(40)) exhibited the downfield shifted P-31 resonance of U(33)pGm(34) indicative of U-turns; ASL(Phe)(Cm-32,Gm(34),m(5)C(40)) did not. An unusual backwards NOE between Gm(34) and A(35) (Gm(34)/H8 to A(35)/H1') was observed in both molecules. The RNAs exhibited a protonated A(38)(+) resulting in the final structures having C-32.A(38)(+) intra-loop base- pairs, With that of ASL (Phe)-(Cn132,Gm(34),m(1)G(37),m(5)C(40)) being especially well defined. A single family of low-energy structures of ASL(Phe)(Cm-32,Gm(34),m(1)G(37),m(5)C(40)) (loop RMSD 0.98 Angstrom) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASL (Phe)-(Cm-32,Gm(34),m(5)C(40)) (loop RMSD 2.58 Angstrom). In addition, the ASL(Phe)- (Cm-32,Gm(34),m(1)G(37),m(1)C(40)) average structure had a greater degree of similarity to that of the yeast tRNA(Phe) crystal structure. A comparison of the resulting structures indicates that modification of position 37 affects the accuracy of decoding and the maintenance of the mRNA reading frame by restricting anticodon loop conformational space. (C) 2003 Elsevier Ltd. All rights reserved.