Structural and functional roles of the N1- and N3-protons of Ψ at tRNA's position 39

Abstract

Pseudouridine at position 39 (Ψ39) of tRNA's anticodon stem and loop domain (ASL) is highly conserved. To determine the physicochemical contributions of Ψ39 to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe) ASL and ASLs with various derivatives of U39 and Ψ39. Ψ39 increased the thermal stability of the ASL (ΔT(m) = 1.3 ± 0.5°C), but did not significantly affect ribosomal binding (K(d) = 229 ± 29 nM) compared to that of the unmodified ASL (K(d) = 197 ± 58 nM). The ASL-Ψ39 P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL. The stability, ribosome binding and fingerprint of the ASL with m1Ψ39 were comparable to that of the ASL with Ψ39. Thus, the contribution of Ψ39 to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U. In contrast, substitutions of m3Ψ39, the isosteric m3U39 and m1m3Φ39 destabilized the ASL by disrupting the A31-U39 base pair in the stem, as confirmed by NMR. N3-methylations of both U and Ψ dramatically decreased ribosomal binding (K(d) = 1060 ± 189 to 1283 ± 258 nM). Thus, canonical base pairing of Ψ39 to A31 through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U39 to Ψ39 may have biological roles other than tRNA's binding to the ribosomal P-site.