Sperger J M, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Biochemistry. 2001 Jun 19;40(24):7005-16. doi: 10.1021/bi0103359.
The ribonucleoprotein enzyme telomerase adds telomeric repeats to the ends of linear chromosomes. The Tetrahymena telomerase reverse transcriptase (TERT) protein and the telomerase RNA can be reconstituted into an active complex in vitro in rabbit reticulocyte lysates. We have probed the structure of the telomerase RNA in the reconstituted complex with RNases T1 and V1. Upon TERT binding to the RNA, sites of both protection and enhancement of cleavage were observed, suggesting potential protein-binding sites and conformational changes in the RNA. Especially prominent was a large region of RNase V1 protection in stem-loop IV. A number of loop IV mutants still bound TERT but showed drastic decreases in the level of telomerase activity and the loss of protein-dependent folding of the pseudoknot region of the telomerase RNA. The telomerase activity defect and the misfolding of the pseudoknot were partially separable, leading to the proposal of two functions for stem-loop IV: to aid in the folding of the pseudoknot and to function more directly in the active site of telomerase. Thus an RNA element far from the template makes a major contribution to Tetrahymena telomerase enzyme activity.
核糖核蛋白酶端粒酶将端粒重复序列添加到线性染色体的末端。嗜热四膜虫端粒酶逆转录酶（TERT）蛋白和端粒酶RNA可以在体外兔网织红细胞裂解物中重组为活性复合物。我们用核糖核酸酶T1和V1探测了重组复合物中端粒酶RNA的结构。在TERT与RNA结合后，观察到了切割受保护和增强的位点，这表明RNA中存在潜在的蛋白质结合位点和构象变化。特别显著的是茎环IV中有一大片核糖核酸酶V1保护区域。许多环IV突变体仍然能结合TERT，但端粒酶活性水平大幅下降，端粒酶RNA假结区域的蛋白质依赖性折叠也丧失了。端粒酶活性缺陷和假结错误折叠部分可分离，这导致人们提出茎环IV的两种功能：帮助假结折叠，并在端粒酶活性位点更直接地发挥作用。因此，一个远离模板的RNA元件对嗜热四膜虫端粒酶的酶活性有重大贡献。
