对来自嗜热四膜虫的端粒酶与模型端粒DNA结合的电子显微镜观察。

Electron microscopic visualization of telomerase from Euplotes aediculatus bound to a model telomere DNA.

作者信息

Fouché Nicole, Moon Ian K, Keppler Brian R, Griffith Jack D, Jarstfer Michael B

机构信息

Lineberger Comprehensive Cancer Center and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

出版信息

Biochemistry. 2006 Aug 8;45(31):9624-31. doi: 10.1021/bi060313s.

DOI:10.1021/bi060313s

PMID:16878997

Abstract

Binding of the telomerase ribonucleoprotein from the ciliate Euplotes aediculatus to telomeric DNA in vitro has been examined by electron microscopy (EM). Visualization of the structures that formed revealed a globular protein complex that localized to the DNA end containing the E. aediculatus telomere consensus 3'-single-strand T(4)G(4)T(4)G(4)T(4)G(2) overhang. Gel filtration confirmed that purified E. aediculatus telomerase is an active dimer in solution, and comparison of the size of the DNA-associated complex with apoferritin suggests that E. aediculatus telomerase binds to a single telomeric 3'-end as a dimer. Up to 43% of the telomerase-DNA complexes appeared by EM to involve tetramers or larger multimers of telomerase in association with two or more DNA ends. These data provide the first direct evidence that telomerase is a functional dimer and suggest that two telomerase ribonucleoprotein particles cooperate to elongate each Euplotes telomere in vivo.

摘要

通过电子显微镜（EM）检测了来自纤毛虫嗜热栖热放线菌的端粒酶核糖核蛋白与端粒DNA在体外的结合情况。对形成的结构进行观察发现，有一个球状蛋白质复合物定位于含有嗜热栖热放线菌端粒共有序列3'-单链T(4)G(4)T(4)G(4)T(4)G(2)突出端的DNA末端。凝胶过滤证实，纯化的嗜热栖热放线菌端粒酶在溶液中是一种活性二聚体，将与DNA相关的复合物的大小与脱铁铁蛋白进行比较表明，嗜热栖热放线菌端粒酶以二聚体形式结合到单个端粒3'-末端。通过电子显微镜观察，高达43%的端粒酶-DNA复合物似乎涉及端粒酶四聚体或更大的多聚体与两个或更多DNA末端的结合。这些数据提供了首个直接证据，证明端粒酶是一种功能性二聚体，并表明两个端粒酶核糖核蛋白颗粒在体内协同作用以延长每个嗜热栖热放线菌的端粒。