Department of Chemistry and Biochemistry, Molecular Biology Institute, PO Box 951569, University of California, Los Angeles, CA 90095-1569, USA.
Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18761-8. doi: 10.1073/pnas.1013269107. Epub 2010 Oct 21.
Telomerase is a unique ribonucleoprotein complex that catalyzes the addition of telomeric DNA repeats onto the 3' ends of linear chromosomes. All vertebrate telomerase RNAs contain a catalytically essential core domain that includes the template and a pseudoknot with extended helical subdomains. Within these helical regions is an asymmetric 5-nt internal bulge loop (J2a/b) flanked by helices (P2a and P2b) that is highly conserved in its location but not sequence. NMR structure determination reveals that J2a/b forms a defined S-shape and creates an ∼90 ° bend with a surprisingly low twist (∼10 °) between the flanking helices. A search of RNA structures revealed only one other example of a 5-nt bulge, from hepatitis C virus internal ribosome entry site, with a different sequence but the same structure. J2a/b is intrinsically flexible but the interhelical motions across the loop are remarkably restricted. Nucleotide substitutions in J2a/b that affect the bend angle, direction, and interhelical dynamics are correlated with telomerase activity. Based on the structures of P2ab (J2a/b and flanking helices), the conserved region of the pseudoknot (P2b/P3, previously determined) and the remaining helical segment (P2a.1-J2a.1 refined using residual dipolar couplings and the modeling program MC-Sym) we have calculated an NMR-based model of the full-length pseudoknot. The model and dynamics analysis show that J2a/b serves as a dominant structural and dynamical element in defining the overall topology of the core domain, and suggest that interhelical motions in P2ab facilitate nucleotide addition along the template and template translocation.
端粒酶是一种独特的核糖核蛋白复合物，能催化线性染色体 3' 末端添加端粒 DNA 重复序列。所有脊椎动物的端粒酶 RNA 都包含一个催化必需的核心结构域，其中包括模板和一个具有扩展螺旋亚结构域的假结。在这些螺旋区域内，有一个不对称的 5-nt 内部凸起环（J2a/b），由螺旋（P2a 和 P2b）环绕，其位置高度保守，但序列不保守。NMR 结构测定表明，J2a/b 形成一个明确的 S 形，并在侧翼螺旋之间形成一个令人惊讶的低扭曲（约 10°）的约 90°弯曲。对 RNA 结构的搜索仅发现另一个来自丙型肝炎病毒内部核糖体进入位点的 5-nt 凸起的例子，其序列不同，但结构相同。J2a/b 是固有灵活的，但跨环的螺旋间运动受到显著限制。影响弯曲角度、方向和螺旋间动力学的 J2a/b 核苷酸取代与端粒酶活性相关。基于 P2ab（J2a/b 和侧翼螺旋）的结构、假结的保守区域（P2b/P3，先前已确定）和剩余的螺旋段（P2a.1-J2a.1 使用残余偶极耦合和建模程序 MC-Sym 进行细化），我们计算了完整假结的基于 NMR 的模型。该模型和动力学分析表明，J2a/b 作为定义核心结构域整体拓扑的主要结构和动力学元件，表明 P2ab 中的螺旋间运动有助于核苷酸沿模板的添加和模板的易位。
