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 tRNAPhe anticodon stem and loop (ASLPhe) with naturally occurring modified nucleosides differing only at position 37, ASLPhe-(Cm32,Gm34,m5C40), and ASLPhe-(Cm32,Gm34,m1G37,m5C40), were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD∼0.6 Å) 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 U33 H2′ and characteristic of tRNAʹs anticodon domain U-turn were present but weak, whereas those involving the U33 H1′ proton were absent from the spectra of both ASLs. However, ASLPhe-(Cm32,Gm34,m1G37,m5C40) exhibited the downfield shifted 31P resonance of U33pGm34 indicative of U-turns; ASLPhe-(Cm32,Gm34,m5C40) did not. An unusual “backwards” NOE between Gm34 and A35 (Gm34/H8 to A35/H1′) was observed in both molecules. The RNAs exhibited a protonated A+38 resulting in the final structures having C32·A+38 intra-loop base-pairs, with that of ASLPhe-(Cm32,Gm34,m1G37,m5C40) being especially well defined. A single family of low-energy structures of ASLPhe-(Cm32,Gm34, m1G37,m5C40) (loop RMSD 0.98 Å) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASLPhe-(Cm32,Gm34,m5C40) (loop RMSD 2.58 Å). In addition, the ASLPhe-(Cm32,Gm34,m1G37,m5C40) average structure had a greater degree of similarity to that of the yeast tRNAPhe 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.