Tesmer V M, Ford L P, Holt S E, Frank B C, Yi X, Aisner D L, Ouellette M, Shay J W, Wright W E
Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039, USA.
Mol Cell Biol. 1999 Sep;19(9):6207-16. doi: 10.1128/MCB.19.9.6207.
We have mapped the 5' and 3' boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.
我们利用源自兔网织红细胞裂解物和人细胞提取物的体外组装系统，绘制了人类端粒酶RNA（hTR）区域的5'和3'边界，该区域是与人类蛋白质催化亚基（hTERT）产生活性所必需的。451个碱基的hTR中跨越核苷酸+33至+325的区域是产生与全长hTR相当的端粒酶活性水平所需的最小序列。我们的结果表明，模板下游约270个碱基的序列是体外有效组装活性端粒酶所必需的；该序列包含的hTR 3'端部分比以前认为的必要部分大得多。此外，我们鉴定出hTR的两个片段（核苷酸+33至+147和+164至+325），它们分别与hTERT组合时不能产生端粒酶活性，但可以共同发挥作用来组装活性端粒酶。这些结果表明，hTR的最小序列可分为两个部分，这两个部分都是体外从头组装活性端粒酶所必需的。
