Conformational analysis of the telomerase RNA pseudoknot hairpin by Raman spectroscopy.
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 nu(s)(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 (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 = 23.6 +/- 1.6 degrees C for WT and 19.7 +/- 2.8 degrees C for MT; T(m) 2 = 68.9 +/- 1.8 degrees C for WT and 70.9 +/- 1.1 degrees 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.
Reipa
Vytas
V
Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8311, USA.
Niaura
Gediminas
G
Atha
Donald H
DH
D001483:Base Sequence; D019871:Dyskeratosis Congenita / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D020871:RNA Stability; D012265:Ribonucleotides / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D044366:Transition Temperature
D012265:Ribonucleotides; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase