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. 2018 Feb 16;46(3):1525-1540.
doi: 10.1093/nar/gkx1275.

Structure and Function of the N-terminal Domain of the Yeast Telomerase Reverse Transcriptase

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Free PMC article

Structure and Function of the N-terminal Domain of the Yeast Telomerase Reverse Transcriptase

Olga A Petrova et al. Nucleic Acids Res. .
Free PMC article

Abstract

The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

Figures

Figure 1.
Figure 1.
Domain structure of telomerase reverse transcriptase (TERT). The TEN (Telomerase Essential N-terminal domain), TRBD (Telomerase RNA-Binding Domain), RT (Reverse Transcriptase) and CTE (C-Terminal Extension) domains are shown as gray boxes. Conserved sequence motifs are shown schematically in dark gray. Two fragments (1–153 and 179–783) of TERT from H. polymorpha discussed in this paper are shadowed in light blue.
Figure 2.
Figure 2.
Structure of hpTEN. (A) X-ray structure of hpTEN, rainbow-colored from the N- to the C-terminus (missing protein fragments are represented as dashes); (B) NMR structure of hpTEN (a representative conformer); (C) NMR structure of hpTEN (a stereo view of a family of 20 conformers).
Figure 3.
Figure 3.
The comparison of the structures of hpTEN and ttTEN. (A) The structure of hpTEN (red elements are superimposed for hpTEN and ttTEN (α7–α8–Gly–α9); yellow elements partially correspond, white fragments distinctly differ in two structures or are missing in one of them). (B) Crystal structure of ttTEN (PDB code 2B2A) in the same color scheme. (C) Electrostatic surface potential of the hpTEN. (D) Electrostatic surface potential of the ttTEN. The two views of each protein are related by a 180° rotation about the y-axis. Protein orientation in left view is identical to that shown at panels A or B. Labeled are C-terminal tails.
Figure 4.
Figure 4.
Structure-based sequence alignment of the TEN domains. (A) Structure-based sequence alignment of hpTEN (combined NMR and X-ray, residues invisible in X-ray structures are shown in lowercase) with ttTEN (PDB id 2B2A) and hTEN (predicted). Two conserved blocks of sequence motif T2 are shadowed in green, the central loop region is shadowed in orange and conserved residues are shown in bold. Residue numbering corresponds to hpTEN. Numbering of the secondary structure elements correspond to hpTEN and ttTEN. (B–D) Ribbon representation of the structure of hpTEN (B), the predicted structure of hTEN (C) and the structure of ttTEN (D). Structural core elements corresponding to the hpTEN motif α7–α8–Gly–α9 are shown in dark cyan; fragments corresponding to the central loop 71–99 in hpTEN are in orange.
Figure 5.
Figure 5.
Interaction of hpTEN with RNA and/or DNA fragments. (A) Histograms of 1H and 15N chemical shift changes and the structural location of the TEN domain residues that are most affected by interactions with nucleic acid fragments (NMR titration, Table 4). 15N-labeled TEN was titrated by ssRNA (E1); heteroduplex RNA–DNA (E3); heteroduplex RNA–DNA (E4); RNA upstream fragment (E10); consecutive titration of TEN by ssRNA (E5, black bars) followed by ssDNA (E6, blue bars); half-fork RNA–DNA (E9); RNA hairpin (E11); (h) RNA–DNA fork with inverted orientation (E7); RNA–DNA fork with direct (native) orientation (E8). Blue bars represent the interacting residues of cluster I, red bars represent cluster II. (B and C) Structure of the TEN domain. Residues that interact with the heteroduplex E3 (B, cluster I) or the native fork E8 (С, cluster II) are colored according to the chemical shift perturbation (yellow – no interaction, blue or red – maximum change). Residues that are not observed in 1H–15N HSQC spectra are colored white.
Figure 6.
Figure 6.
The proposed role of the TEN domain in the telomerase function. (A) Cartoon of the minimal telomerase complex. (B) Combined 3D hypothetical model of hpTERT (the TERT ring is colored according its TRBD, RT and CTE domains, TEN domain is colored as described in Figure 4C) complexed with the RNA–DNA fork (telomerase RNA in green, telomeric DNA in blue). The view on the left shows TEN domain of hpTERT in the orientation similar to that used in Figures 2A and 3A. The second view, on the right, is related by a 90° rotation about the axis shown.

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