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

Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop do main (ASL) is highly conserved. To determine the physicochemical contributi ons of Psi(39) to the ASL and to relate these properties to tRNA function i n translation, we synthesized the unmodified yeast tRNA(Phe) ASL and ASLs w ith various derivatives of U-39 and Psi(39). Psi(39) increased the thermal stability of the ASL (Delta T-m = 1.3 +/- 0.5 degrees C), but did not signi ficantly affect ribosomal binding (K-d = 229 +/- 29 nM) compared to that of the unmodified ASL (K-d = 197 +/- 58 nM). The ASL-Psi(39) P-site fingerpri nt on the 30S ribosomal subunit was similar to that of the unmodified ASL, The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39) were comparable to that of the ASL with Psi(39). Thus, the contribution of Psi(39) to ASL stability is not related to N1-H hydrogen bonding, but prob ably is due to the nucleoside's ability to improve base stacking compared t o U. In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39) and m(1)m(3)Psi(39) destabilized the ASL by disrupting the A(31)-U-39 base pai r in the stem, as confirmed by NMR, N3-methylations of both U and Psi drama tically decreased ribosomal binding (K-d = 1060 +/- 189 to 1283 +/- 258 nM) . Thus, canonical base pairing of Psi(39) to A(31) through N3-H is importan t to structure, stability and ribosome binding, whereas the increased stabi lity and the N1-proton afforded by modification of U-39 to Psi(39) may have biological roles other than tRNA's binding to the ribosomal P-site.