Structural analysis of the telomerase RNA pseudoknot domain by Raman spectroscopy

We have measured the Raman band intensities and frequencies of two 30‐mer ribonucleotides that represent the wild type and dyskeratosis (DKC) mutant GC (107‐108)→AG structures of the pseudoknot hairpin region of human telomerase RNA. We have used the Raman hypochromism of vibrational bands, previously assigned to specific bases or conformational RNA markers, to observe alterations in the pentaloop and stem structures of these two structures as a function of temperature. We observe that the intense ν (O‐P‐O) band at 812 cm −1 indicates the presence of A‐form backbone structure at relatively low temperatures in both the wild type (WT) and mutant (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. Most likely, one of the rU residues in pentaloop transforms from C2′‐ endo/anti to C3′‐ endo/anti conformation upon mutation. Two transition temperatures (T m ) 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 (T m 1 = 23.5 ± 1.8 o C for WT and 20.2 ± 4.2 o C for MT; T m 2 = 69.5 ± 2.5 o C for WT and 70.6 ± 1.2 o C for MT). From these results we have determined 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 wild type 30‐mer.