Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892
Proc Natl Acad Sci U S A. 2022 Jan 4;119(1). doi: 10.1073/pnas.2116159118.
Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of telomerase reverse transcriptase (TERT) and cryoelectron microscopy (cryo-EM) structures of human and telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using TERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by TERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of TERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that TERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of TERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by TERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.
端粒酶通过从短 RNA 模板进行重复逆转录，在线性染色体的末端合成端粒。端粒酶逆转录酶 (TERT) 的晶体结构和人类端粒的冷冻电镜 (cryo-EM) 结构揭示了逆转录酶结构域中的保守特征，包括靠近 DNA 3'端的腔和与 RNA 模板的紧密相互作用。为了使 RNA 模板进行易位，它需要未配对并与 DNA 产物分离。在这里，我们研究了结构腔容纳环出 DNA 凸起并使 RNA/DNA 杂交体分离的潜力。使用 TERT 作为模型系统，我们表明 DNA 引物中的环出端粒重复序列可以被 TERT 容纳和延伸，但不能被逆转录病毒逆转录酶容纳和延伸。减小腔尺寸的突变会降低 TERT 延伸环出 DNA 底物的能力。与端粒酶的 cryo-EM 结构一致，我们发现 TERT 需要在 RNA 模板和 DNA 引物之间至少有 4 个碱基才能有效地进行 DNA 合成。我们还确定了 TERT 的三元复合物结构，包括在 2.0-Å 分辨率下的下游 RNA/DNA 杂交体，并表明下游 RNA 双链体，相当于端粒酶 RNA 中的 5'模板结合元件，可增强 TERT 进行端粒合成的效率。尽管 TERT 具有无需开-合构象变化即可形成的预形成活性位点，但它包含容纳环出 RNA 和 DNA 的腔。灵活的 RNA-DNA 结合可能是端粒重复添加过程的基础。
