Solution Structure and Dynamics of the Wild-type Pseudoknot of Human Telomerase RNA
Solution Structure and Dynamics of the Wild-type Pseudoknot of Human Telomerase RNA
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DOI：
10.1016/j.jmb.2008.10.005
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发表时间：
2008-12-31
影响因子：
5.6
通讯作者：
Feigon, Juli
中科院分区：
文献类型：
--
作者：
Kim, Nak-Kyoon;Zhang, Qa;Feigon, Juli
端粒酶是一种核糖核蛋白复合物，通过连续添加端粒重复 DNA 来复制线性染色体的 3' 末端。端粒酶全酶包含两个必需的催化成分：端粒酶逆转录酶 (TERT) 和端粒酶 RNA (TER)。 TER 包括端粒重复合成的模板以及功能所需的其他域。我们报告了用大量 RDC 精制的野生型最小保守人 TER 假结的溶液结构，并详细分析了凸起 U177 对假结结构的影响、通过 RDC 和 C-13 弛豫测量分析的动力学以及碱基对稳定性。 PKWT 的整体结构与之前报道的 Delta U177 假结 (PKDU) 高度相似，后者删除了对催化活性很重要的保守凸起 U。为了与 PKWT 进行直接比较，PKDU 的结构使用一组可比较的 RDC 进行了重新细化。两个假结在两个茎的连接处都含有催化必需的三螺旋，包括两个茎1-环2小沟三联体、一个连接环1-环2 Hoogsteen碱基对和位于凸起U177正上方的茎2-环1大沟U.A-U Watson-Crick-Hoogsteen三联体。然而，凸起附近的碱基对的稳定性和某些核苷酸的动力学存在显着差异。 U177凸起周围的茎2碱基对的稳定性大大降低，导致三螺旋中的Watson-Crick对在Hoogsteen对之前开始展开，这可能会影响端粒酶的组装和活性。凸起 U 位于与三螺旋相互作用相反的面上的小凹槽中，并在空间上阻断 A176 2'OH，最近有人提出 A176 2'OH 在催化中发挥作用。 U形凸起可充当铰链，在该区域提供脊柱灵活性。 (C) 2008 Elsevier Ltd. 保留所有权利。
Telomerase is a ribonucleoprotein complex that replicates the 3' ends of linear chromosomes by successive additions of telomere repeat DNA. The telomerase holoenzyme contains two essential components for catalysis, a telomerase reverse transcriptase (TERT) and telomerase RNA (TER). The TER includes a template for telomere repeat synthesis as well as other domains required for function. We report the solution structure of the wildtype minimal conserved human TER pseudoknot refined with an extensive set of RDCs, and a detailed analysis of the effect of the bulge U177 on pseudoknot structure, dynamics analyzed by RDC and C-13 relaxation measurements, and base pair stability. The overall structure of PKWT is highly similar to the previously reported Delta U177 pseudoknot (PKDU) that has a deletion of a conserved bulge U important for catalytic activity. For direct comparison to PKWT, the structure of PKDU was re-refined with a comparable set of RDCs. Both pseudoknots contain a catalytically essential triple helix at the junction of the two stems, including two stem 1-loop 2 minor groove triples, a junction loop 1-loop 2 Hoogsteen base pair, and stem 2-loop 1 major groove U.A-U Watson-Crick-Hoogsteen triples located directly above the bulge U177. However, there are significant differences in the stabilities of base pairs near the bulge and the dynamics of some nucleotides. The stability of the base pairs hi stem 2 surrounding the bulge U177 is greatly decreased, with the result that the Watson-Crick pairs in the triple helix begin to unfold before the Hoogsteen pairs, which may affect telomerase assembly and activity. The bulge U is positioned in the minor groove on the face opposite the triple helical interactions, and sterically blocks the A176 2'OH, which has recently been proposed to have a role in catalysis. The bulge U may serve as a hinge providing backbone flexibility in this region. (C) 2008 Elsevier Ltd. All rights reserved.