Structure and folding of theTetrahymenatelomerase RNA pseudoknot

Abstract

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.

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis

“…Also, it is unintuitive that a minimally base-paired structure such as proposed for hTR alt-P3 previously (36) and here could, with a single additional base-pair, present a major challenge for folding of active RNP. Nonetheless, our results support these conclusions.…”

“…As precedent, the t/PK domain of Saccharomyces cerevisiae TER has two conformations with alternative stem pairing (55) as does the Tetrahymena TER t/PK (36,37). The evidence presented in this work for an alt-P3 hTR conformation extends previous studies in yeast and ciliate systems that together suggest a potentially fundamental principle for co-folding of TER and TERT.…”

Structural biology of telomerase and its interaction at telomeres

“…These interactions, including several major groove U-A-U triples, have been shown to be important for activity in human and yeast telomerase [42,43]. The solution NMR structure of the Tetrahymena pseudoknot, which has a much shorter stem 2 than yeasts and vertebrates, was more recently reported [13,19]. The free Tetrahymena pseudoknot is much less stable than those of yeasts and vertebrates, and includes only two major groove triples: a U-A-U and a novel C-G-A.…”

“…Solution NMR studies of the entire t/PK as well as smaller RNA constructs provided additional details of the t/PK secondary structure and determined that the entire pseudoknot sequence is sequestered in an alternative helical structure that includes the template nucleotides (Figure 3a) [19]. This free TER structure has both of the high affinity TERT TRBD binding sites exposed (TBE and STE/loop 4); the authors propose that TERT assembly with TER could be initiated at the TBE.…”

“…Somewhat more rapid structural progress had been made with telomere binding proteins, especially from yeasts [12]. During the past couple of years, a surge of progress has been made on structural biology of telomerase, including reports of the first cryo-electron microscopy (cryo-EM) structure of telomerase, from Tetrahymena [13], crystal structures of TERT and TERT-interacting protein domains [14–17], telomerase RNA structures and models [18,19], and identification in Tetrahymena telomerase holoenzyme of human homologues of telomere-associated proteins [13,20] that have provided a more unified view of telomerase interaction at telomeres [21]. Here we review these and other studies published since 2015 on structural investigations of primarily Tetrahymena and vertebrate telomerase and its interaction at telomeres.…”