Stabilization of the anticodon stem-loop of tRNALys,3 by an A+-C base-pair and by pseudouridine

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNALys,3. The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters derived from temperature dependent UV measurements. The pseudouridine-containing hairpin is thermodynamically more stable than the unmodified hairpin by 5 degreesC, and this corresponds with increased base stacking on the 3' side of the tRNA anticodon loop. An A+38-C32 base-pair also forms at the base of the anticodon stem with an approximate pKa of 6 for A38. Formation of the A+-C base-pair increases the Tm of both pseudouridine modified and unmodified RNA hairpins by 5-6 degreesC, and decreases the DeltaG degrees for hairpin formation by 1 kcal/mol. Solution structures were determined for both psi39 and unmodified hairpins under limiting pH conditions at pH 5 and pH 7 to assess the structural effects of both psi modification and the additional A+-C base-pair on tRNALys,3 structure. The A+38-C32 base-pair strengthens the 31-39 base-pair, and induces formation of a dynamic U33-A37 base-pair that effectively reduces the normal seven nucleotide anticodon loop to a three nucleotide UUU loop. These undermodified tRNALys,3 anticodon loops are distinctly different from those seen for other tRNAs exemplified by tRNAPhe. The conformation of the tRNA loop has important implications for the role of nucleoside modification in codon-anticodon recognition and for utilization of tRNALys,3 by HIV-1 as the native reverse transcriptase primer.