Reipa Vytas, Niaura Gediminas, Atha Donald H
Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8311, USA.
RNA. 2007 Jan;13(1):108-15. doi: 10.1261/rna.182607. Epub 2006 Nov 21.
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.
我们测量了两种30聚体核糖核苷酸的温度依赖性拉曼光谱，它们分别代表人类端粒酶RNA假结发夹区域的野生型（WT）和先天性角化不良（DKC）突变型（MT）GC（107 - 108）→AG结构。我们将这些先前通过紫外熔解和核磁共振表征的结构用作拉曼研究的模型系统。我们观察到，先前归属于特定碱基或构象RNA标记的振动带的拉曼减色现象，反映了这两种寡核苷酸的五聚环和茎结构中与温度相关的变化。我们还观察到，在812 cm⁻¹处强烈的ν(s)(O - P - O)带表明在WT和MT RNA序列中相对较低温度下存在A形式的主链结构。该突变导致五聚环中与C2'-内/反式核糖构象相关的1244 cm⁻¹处尿苷（rU）带强度降低。通过分析与胞苷（rC）残基振动相关的1256 cm⁻¹带的拉曼差异强度 - 温度曲线，确定了两个转变温度（T(m)），特别是C2'-内/反式核糖构象（WT的T(m) 1 = 23.6 ± 1.6℃，MT的T(m) 1 = 19.7 ± 2.8℃；WT的T(m) 2 = 68.9 ± 1.8℃，MT的T(m) 2 = 70.9 ± 1.1℃）。从这些结果我们可以得出结论，DKC突变型30聚体在五聚环区域表现出比WT 30聚体更低的稳定性，而在茎区域表现出略高的稳定性。这表明先前归属于特定碱基或构象RNA标记的拉曼带可用于探测端粒酶假结发夹序列的局部结构特征。
