We have measured the Raman band intensities and frequencies of two 30-mer ribonucleotides that represent the wild type (WT) and dyskeratosis 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 n(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 and mutant (MT) RNA sequences. The mutation induces a decrease in the intensity of the 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. From the analysis of the Raman difference intensity temperature profiles of the n(O-P-O) band the transition temperatures for the melting of A-form stem structures were determined to be 55.9 4.1 and 51.6 1.1 oC for WT and MT RNA, respectively. From the analysis of intensity-temperature profile of 1230-cm-1 band it was found that the melting temperature associated with stacking interaction of rU residues is slightly higher for WT (47.3 0.7 oC) as compared with MT (44.2 0.6 oC) sample. Two transition temperatures were determined from the analysis of Raman difference intensity-temperature profiles of 1256-cm-1 band associated with vibrations of rC residues, in particular the C2 -endo/anti ribose conformation. From these results we have determined that the DKC mutant 30-mer exhibits a lower stability in the pentaloop region than the wild type 30-mer. These results demonstrate that Raman band intensities and frequencies, previously assigned to specific bases or conformational RNA markers, can be used to probe structural features of the telomerase pseudoknot structure in order to evaluate current functional models.
Citation: Faseb Journal
Issue: No. 5
Pub Type: Journals
pseudoknot, Raman spectroscopy, RNA, telomerase