Gilley D, Blackburn E H
Department of Microbiology and Immunology, University of California, San Francisco 94143-0414, USA.
Mol Cell Biol. 1996 Jan;16(1):66-75. doi: 10.1128/MCB.16.1.66.
The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.
核糖核蛋白酶端粒酶是一种特殊的逆转录酶，它通过复制端粒酶RNA中的模板序列来合成端粒DNA。在这里，我们分析了在体内与突变或野生型端粒酶RNA组装的嗜热四膜虫端粒酶的作用，以进一步确定参与基本酶功能（模板化、底物排列和聚合促进）的特定端粒酶RNA残基的作用。端粒酶RNA的第49位定义了3'模板残基边界，表明43至49位的七个位置能够作为模板残基。我们直接证明了引物底物的定位涉及引物与端粒酶RNA残基之间的沃森-克里克碱基配对。出乎意料的是，引物DNA与端粒酶RNA残基50之间特异性形成沃森-克里克碱基对对于促进引物延伸至关重要。相比之下，将第49位的胞嘧啶突变为G的突变端粒酶表现出高效的3'错配延伸。这项工作为特定的引物-端粒酶相互作用以及涉及端粒酶RNA的碱基特异性相互作用在端粒酶的基本活性位点功能中发挥作用提供了新证据。
