Zappulla David C, Goodrich Karen, Cech Thomas R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Nat Struct Mol Biol. 2005 Dec;12(12):1072-7. doi: 10.1038/nsmb1019. Epub 2005 Nov 20.
The ribonucleoprotein enzyme telomerase synthesizes DNA at the ends of chromosomes. Although the telomerase catalytic protein subunit (TERT) is well conserved, the RNA component is rapidly evolving in both size and sequence. Here, we reduce the 1,157-nucleotide (nt) Saccharomyces cerevisiae TLC1 RNA to a size smaller than the 451-nt human RNA while retaining function in vivo. We conclude that long protein-binding arms are not essential for the RNA to serve its scaffolding function. Although viable, cells expressing Mini-T have shortened telomeres and reduced fitness as compared to wild-type cells, suggesting why the larger RNA has evolved. Previous attempts to reconstitute telomerase activity in vitro using TLC1 and yeast TERT (Est2p) have been unsuccessful. We find that substitution of Mini-T for wild-type TLC1 in a reconstituted system yields robust activity, allowing the contributions of individual yeast telomerase components to be directly assessed.
核糖核蛋白酶端粒酶在染色体末端合成DNA。尽管端粒酶催化蛋白亚基（TERT）高度保守，但RNA组分在大小和序列上都在快速进化。在此，我们将1157个核苷酸（nt）的酿酒酵母TLC1 RNA缩减至比451 nt的人类RNA更小的尺寸，同时在体内保留其功能。我们得出结论，长的蛋白质结合臂对于RNA发挥其支架功能并非必不可少。虽然表达Mini-T的细胞是存活的，但与野生型细胞相比，其端粒缩短且适应性降低，这表明了较大RNA进化的原因。先前使用TLC1和酵母TERT（Est2p）在体外重建端粒酶活性的尝试均未成功。我们发现，在重组系统中用Mini-T替代野生型TLC1可产生强大的活性，从而能够直接评估单个酵母端粒酶组分的作用。
