期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：1
DOI：10.1073/pnas.2116159118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Telomerase reverse transcriptase (TERT) has a conserved central cavity near the active site. Using enzymatic and mutagenesis analyses, we provide experimental evidence that an artificially looped-out telomeric repeat in the DNA primer can be transiently accommodated in the cavity of
Tribolium castaneum (
tcTERT). Kinetically,
tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. An RNA duplex downstream of the RNA-template region after a flexible linker enhances the efficiency of primer extension by
tcTERT. In addition to the peripheral cavities that accommodate looped-out RNA during each telomeric repeat synthesis, the central cavity that can accommodate the looped-out DNA may aid RNA-template translocation between cycles of telomeric repeat synthesis.
Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of
Tribolium castaneum telomerase reverse transcriptase (
tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and
Tetrahymena 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
tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by
tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of
tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that
tcTERT 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
tcTERT 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
tcTERT. 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.
