Folding heterogeneity in the essential human telomerase RNA three-way junction.
Autor: | Palka C; Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA., Forino NM; Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064, USA., Hentschel J; Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA., Das R; Biophysics Program, Stanford University, Stanford, California 94305, USA.; Department of Biochemistry, Stanford University, Stanford, California 94305, USA.; Department of Physics, Stanford University, Stanford, California 94305, USA., Stone MD; Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.; Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA. |
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Jazyk: | angličtina |
Zdroj: | RNA (New York, N.Y.) [RNA] 2020 Dec; Vol. 26 (12), pp. 1787-1800. Date of Electronic Publication: 2020 Aug 19. |
DOI: | 10.1261/rna.077255.120 |
Abstrakt: | 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. (© 2020 Palka et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.) |
Databáze: | MEDLINE |
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