Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
RNA. 2011 Feb;17(2):298-311. doi: 10.1261/rna.2483611. Epub 2010 Dec 21.
Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.
与具有高度有序整体结构的核糖核蛋白复合物（如核糖体）不同，酵母端粒酶似乎受到的约束要小得多。在这里，我们研究了 Ku 亚基在 1157 个核苷酸的酵母端粒酶 RNA（TLC1）内定位的重要性。TLC1 RNA 中 Ku 结合发夹的 48 个核苷酸缺失（tlc1Δ48）会减少端粒长度、具有大染色体重排的细胞的存活率以及在断裂染色体末端的新端粒添加。为了测试 Ku 在端粒酶 RNP 新位置的功能，我们将其结合位点重新引入到 tlc1Δ48 RNA 的 446 或 1029 位。我们发现 Ku 在体内结合这些重新定位的位点，端粒长度略有增加，但具有统计学意义。端粒酶通过修复受损染色体臂来促进具有大染色体重排的细胞存活的能力也部分恢复，而特定染色体断裂处 DNA 添加的动力学被延迟。在 TLC1 中有两个 Ku 位点会导致可变数量的端粒逐渐过度延长，这与 Ku 在端粒酶招募到染色体末端中的作用一致。TLC1 中的 Ku 结合位点数量有助于体内端粒酶 RNA 的丰度，但仅部分负责端粒长度表型。因此，端粒酶 RNA 水平和端粒长度的调节可以通过端粒酶 RNA 中 Ku 位点的数量来调节。此外，对于天然端粒长度维持，Ku 在端粒酶 RNP 中的相对定位具有很大的灵活性，尽管不如对必需的 Est1p 亚基那样灵活。
