Conformational analysis of the telomerase RNA pseudoknot hairpin by Raman spectroscopy

Abstract

We have measured the temperature-dependent Raman spectra of two 30-mer ribonucleotides that represent the wild-type (WT) and dyskeratosis congenita (DKC) mutant (MT) GC (107–108) → AG structures of the pseudoknot hairpin region of human telomerase RNA. We have used these structures, previously characterized by UV-melting and NMR, as a model system for our Raman investigation. We observe that Raman hypochromism of vibrational bands, previously assigned to specific bases or conformational RNA markers, reflect temperature-dependent alterations in the pentaloop and stem structures of these two oligonucleotides. We also observe that the intense νs(O-P-O) band at 812 cm−1 indicates the presence of A-form backbone structure at relatively low temperatures in both the WT and MT RNA sequences. The mutation induces a decrease in the intensity of the uridine (rU) band at 1244 cm−1 associated with C2′-endo/anti ribose conformation in the pentaloop. Two transition temperatures (Tm) were determined from the analysis of Raman difference intensity-temperature profiles of the 1256 cm−1 band, which is associated with vibrations of cytidine (rC) residues, in particular, the C2′-endo/anti ribose conformation (Tm1 = 23.6 ± 1.6°C for WT and 19.7 ± 2.8°C for MT; Tm2 = 68.9 ± 1.8°C for WT and 70.9 ± 1.1°C for MT). From these results we can conclude that the DKC mutant 30-mer exhibits a lower stability in the pentaloop region and a slightly higher stability in the stem region than the WT 30-mer. This demonstrates that Raman bands, previously assigned to specific bases or conformational RNA markers, can be used to probe local structural features of the telomerase pseudoknot hairpin sequence.