Folding heterogeneity in the essential human telomerase RNA three-way junction
- Christina Palka1,7,
- Nicholas M. Forino2,7,
- Jendrik Hentschel1,
- Rhiju Das3,4,5 and
- Michael D. Stone1,6
- 1Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA
- 2Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064, USA
- 3Biophysics Program, Stanford University, Stanford, California 94305, USA
- 4Department of Biochemistry, Stanford University, Stanford, California 94305, USA
- 5Department of Physics, Stanford University, Stanford, California 94305, USA
- 6Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA
- Corresponding author: mds{at}ucsc.edu
-
↵7 These authors contributed equally to this work.
Abstract
Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. To compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (TWJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, secondary structural modeling, and single-molecule structural analysis. Our data suggest the essential P6.1 stem–loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. Finally, single-molecule FRET measurements of CR4/5 and a mutant designed to stabilize the P6.1 stem demonstrate that TERT binding selects for a structural conformation of CR4/5 that is not the dominant state of the TERT-free in vitro RNA ensemble.
Keywords
Footnotes
-
Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.077255.120.
-
Freely available online through the RNA Open Access option.
- Received July 12, 2020.
- Accepted July 29, 2020.
This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
