Flexibility of telomerase in binding the RNA template and DNA telomeric repeat

• Published in: Proceedings of the National Academy of Sciences - PNAS 2022-01, Vol.119 (1)
• Main Authors: Choi, Woo Suk, Weng, Peter J, Yang, Wei
• Format: Article
• Language: English
• Subjects: Animals; Binding; Biological Sciences; Chromosomes; Crystal structure; Deoxyribonucleic acid; DNA; DNA - genetics; DNA biosynthesis; Holes; Mutation; Protein Binding; Reverse transcription; Ribonucleic acid; RNA; RNA - metabolism; RNA-directed DNA polymerase; Substrates; Synthesis; Telomerase; Telomerase - metabolism; Telomerase reverse transcriptase; Telomere; Telomeres; Templates, Genetic; Tribolium - metabolism
• ISSN: 0027-8424; 1091-6490
• 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.