An RNA Model System for Investigation of Pseudouridine Stabilization of the Codon-Anticodon Interaction in tRNALys, tRNAHisand tRNATyr

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Abstract
The nucleoside conformation of pseudouridine (ψ) was investigated in a series of RNA oligonucleotides and compared with the same sequences containing the parent, unmodified uridine nucleoside. 1H NMR spectroscopy was used to determine the glycosyl conformational preference in pseudouridine systems at the nucleoside level; these experiments were extended to trimers, and ultimately to RNA tetraloop hairpins that are models for the codonanticodon interaction in tRNA. ROESY 1D and 2D NMR experiments were used to measure the nucleoside conformational preference as a function of temperature. The thermodynamic stability of the RNA tetraloops was also analyzed using UV monitored Tm experiments which established that pseudouridine has a very strong stabilizing effect on double- stranded, base pairing interactions when the modification is located within a base-paired region. This was shown for a tetraloop hairpin model of the codon-anticodon interaction in tRNATyr) which contains a ψ at position 35. Pseudouridine also stabilizes double-stranded RNA when the ψ modification is in a single-stranded region adjacent to a duplex region as occurs for y at positions 38 or 39 in tRNALys and tRNAHis. These results establish that pseudouridine modification of RNA is a powerful and versatile mechanism for stabilizing local RNA structure in both single-stranded and double-stranded regions. Previously postulated roles for pseudouridine as a “conformational switch” are unlikely in light of the increased barrier to rotation about the glycosyl bond upon modification of uridine to pseudouridine. The Tm and NMR data show that local RNA stacking stabilization as a result of y will stabilize adjacent double-stranded RNA regions such as the codon-anticodon interaction in tRNA.