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Title: Structure and folding of the Tetrahymena telomerase RNA pseudoknot

Telomerase maintains telomere length at the ends of linear chromosomes using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). An essential part of TER is the template/pseudoknot domain (t/PK) which includes the template, for adding telomeric repeats, template boundary element (TBE), and pseudoknot, enclosed in a circle by stem 1. The Tetrahymena telomerase holoenzyme catalytic core (p65-TER-TERT) was recently modeled in our 9 Å resolution cryo-electron microscopy map by fitting protein and TER domains, including a solution NMR structure of the Tetrahymena pseudoknot. Here, we describe in detail the structure and folding of the isolated pseudoknot, which forms a compact structure with major groove U•A-U and novel C•G-A + base triples. Base substitutions that disrupt the base triples reduce telomerase activity in vitro. NMR studies also reveal that the pseudoknot does not form in the context of full-length TER in the absence of TERT, due to formation of a competing structure that sequesters pseudoknot residues. The residues around the TBE remain unpaired, potentially providing access by TERT to this high affinity binding site during an early step in TERT-TER assembly. A model for the assembly pathway of the catalytic core is proposed.
Authors:
 [1] ;  [1]
  1. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry. Molecular Biology Inst.
Publication Date:
Grant/Contract Number:
FC03-02ER63421; GM048123; GM007185; MCB1022379
Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 45; Journal Issue: 1; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Research Org:
Univ. of California, Los Angeles, CA (United States)
Sponsoring Org:
USDOE; National Inst. of Health (NIH) (United States); National Science Foundation (NSF)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; catalytic domain; telomerase; tetrahymena; RNA
OSTI Identifier:
1361669

Cash, Darian D., and Feigon, Juli. Structure and folding of the Tetrahymena telomerase RNA pseudoknot. United States: N. p., Web. doi:10.1093/nar/gkw1153.
Cash, Darian D., & Feigon, Juli. Structure and folding of the Tetrahymena telomerase RNA pseudoknot. United States. doi:10.1093/nar/gkw1153.
Cash, Darian D., and Feigon, Juli. 2016. "Structure and folding of the Tetrahymena telomerase RNA pseudoknot". United States. doi:10.1093/nar/gkw1153. https://www.osti.gov/servlets/purl/1361669.
@article{osti_1361669,
title = {Structure and folding of the Tetrahymena telomerase RNA pseudoknot},
author = {Cash, Darian D. and Feigon, Juli},
abstractNote = {Telomerase maintains telomere length at the ends of linear chromosomes using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). An essential part of TER is the template/pseudoknot domain (t/PK) which includes the template, for adding telomeric repeats, template boundary element (TBE), and pseudoknot, enclosed in a circle by stem 1. The Tetrahymena telomerase holoenzyme catalytic core (p65-TER-TERT) was recently modeled in our 9 Å resolution cryo-electron microscopy map by fitting protein and TER domains, including a solution NMR structure of the Tetrahymena pseudoknot. Here, we describe in detail the structure and folding of the isolated pseudoknot, which forms a compact structure with major groove U•A-U and novel C•G-A+ base triples. Base substitutions that disrupt the base triples reduce telomerase activity in vitro. NMR studies also reveal that the pseudoknot does not form in the context of full-length TER in the absence of TERT, due to formation of a competing structure that sequesters pseudoknot residues. The residues around the TBE remain unpaired, potentially providing access by TERT to this high affinity binding site during an early step in TERT-TER assembly. A model for the assembly pathway of the catalytic core is proposed.},
doi = {10.1093/nar/gkw1153},
journal = {Nucleic Acids Research},
number = 1,
volume = 45,
place = {United States},
year = {2016},
month = {11}
}