Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Nucleic Acids Res. 2010 Jan;38(3):e16. doi: 10.1093/nar/gkp1033. Epub 2009 Nov 17.
The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures.
端粒酶核糖核蛋白酶的活性对于维持基因组稳定性和正常细胞发育至关重要。尽管端粒酶活性具有重要的生物医学意义，但该酶的详细结构模型仍有待建立。在这里，我们报告了一种用于直接结构分析催化活性端粒酶的单分子测定法。在该测定中，寡核苷酸杂交用于探测单个端粒酶酶的引物延伸活性，具有单核苷酸敏感性，从而可以精确区分无活性、有活性和连续结合事件。然后，从酶分子在活性和连续结合事件期间的 FRET 信号用于确定催化活性全酶中端粒酶 RNA 的整体组织。使用该测定法，我们已经在具有不同结构的酶的异质群体中鉴定了端粒酶的一种活性构象。
