Parks Joseph W, Kappel Kalli, Das Rhiju, Stone Michael D
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
RNA. 2017 Feb;23(2):175-188. doi: 10.1261/rna.058743.116. Epub 2016 Nov 15.
Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation.
端粒酶对端粒的维持使得包括大多数人类癌症在内的快速分裂细胞能够持续增殖。尽管其具有直接的生物医学意义，但人类端粒酶复合物的结构仍然未知。生成均匀的端粒酶样本一直是开发改进结构模型的重大障碍。在这里，我们将单分子荧光共振能量转移（smFRET）测量与Rosetta建模相结合，以绘制活性核糖核蛋白内必需的端粒酶RNA核心结构域的构象。FRET引导的建模将必需的假结折叠定位在由C端元件组成的蛋白质表面上活性位点的远端，该结构域与典型的聚合酶拇指结构域具有结构同源性。独立解析的嗜热四膜虫端粒酶的中等分辨率结构对我们的建模方法进行了盲测，并揭示了该结构域在不同生物体中的结构同源性。我们的smFRET-Rosetta模型揭示了催化过程中RNA核心结构域内纳米级的重排。综合来看，我们的FRET数据和准原子分子模型使我们能够提出一种可能的机制，说明RNA核心结构域重排如何与模板杂交延伸相耦合。
