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Structural basis of template-boundary definition in Tetrahymena telomerase


Telomerase is required to maintain repetitive G-rich telomeric DNA sequences at chromosome ends. To do so, the telomerase reverse transcriptase (TERT) subunit reiteratively uses a small region of the integral telomerase RNA (TER) as a template. An essential feature of telomerase catalysis is the strict definition of the template boundary to determine the precise TER nucleotides to be reverse transcribed by TERT. We report the 3-Å crystal structure of the Tetrahymena TERT RNA-binding domain (tTRBD) bound to the template boundary element (TBE) of TER. tTRBD is wedged into the base of the TBE RNA stem-loop, and each of the flanking RNA strands wraps around opposite sides of the protein domain. The structure illustrates how the tTRBD establishes the template boundary by positioning the TBE at the correct distance from the TERT active site to prohibit copying of nontemplate nucleotides.

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Figure 1: T. thermophila telomerase composition and catalytic cycle.
Figure 2: Structure of the tTRBD–TBE complex.
Figure 3: Protein-protein interactions between conserved tTERT RBD motifs.
Figure 4: Protein-RNA interactions within the tTRBD–TBE complex.
Figure 5: Conservation and function of observed interactions in the tTRBD–TBE complex.
Figure 6: Structural model of T. thermophila telomerase RNA-template connectivity to the TER TBE.

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We thank the laboratory of K. Collins (University of California, Berkeley) for the original tTRBD expression plasmids. We thank L. Lancaster and S. Tripathy for technical help with crystallization and data analysis. We thank H. Noller, W. Scott, and members of S. Rubin's laboratory for helpful discussions and technical advice. This work was supported by grants from the US National Institutes of Health (2T32GM008646-16 to L.I.J.) and (RO1GM095850 to M.D.S.)

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Authors and Affiliations



L.I.J., B.M.A., A.O., C.L., S.M.R. and M.D.S. designed the experiments. L.I.J., B.M.A., S.M.R. and M.D.S. wrote the manuscript.

Corresponding author

Correspondence to Michael D Stone.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cross-species TERT RBD sequence alignments.

The ciliate-specific CP2 motif is shown for Tetrahymena only (purple). The conserved CP- and T-motifs are shown in orange and blue, respectively.

Supplementary Figure 2 Ciliate TERT RBD sequence alignments.

The ciliate specific CP2 motif is shown in purple. The conserved CP- and T-motifs are shown in orange and blue, respectively.

Supplementary Figure 3 RNA binding activity of new tTRBD protein construct.

(a) Electrophoretic Mobility Shift Assay (EMSA) of wild type (WT) TER (left panel) and A22U TER (right panel) bound to RBD. (b) Primer extension assay of WT and A22U TER in the presence or absence of dATP. Numbers on the left indicate number of nucleotides extended of the primer. A defect in template boundary definition would be expected to arise in the presence of ATP since TER nucleotide U42 would enter the TERT active site as has been shown previously (Lai, C.K. et al., Genes Dev. 16, 415-20, 2002) (Akiyama, B.M. et al., J Biol Chem. 288, 22141-9, 2013). (c) FPLC elution profiles of the TBE RNA construct alone (top panel) and of the tTRBD-TBE complex (bottom panel). Red and black lines indicate absorbance measured at 260nm and 280nm, respectively. The tTRBD-TBE complex elutes as a single peak around 15.5 ml and free TBE RNA elutes around 17.4 ml. The samples were run through a superdex 200 column.

Supplementary Figure 4 Crystal-packing arrangement of the tTRBD–TBE RNA complex.

(a) There are two protein-RNA complexes in the asymmetric unit. Complex A (light green, cyan and black) is better fit to the electron density than complex B (dark green, teal and grey) and is therefore the molecule chosen for structure determination. (b) Crystal contacts are mediated by RNA-RNA base stacking between complex B and complex A of the neighboring asymmetric unit. This interaction is further highlighted in c (dashed red box). (c) Base stacking interactions between complex B and complex A of the neighboring asymmetric unit. Residues A40 and U41 of complex B are twisted outward compared to the same residues in complex A to mediate crystal contacts with the distal region of stem II of complex A.

Supplementary Figure 5 Comparison of TER secondary structure.

The left panel shows the secondary structure of TBE-Stem II as determined previously (McCormick-Graham, M. et al., Nucleic Acids Res. 23, 1091-7, 1995). The right panel shows the updated TER secondary structure based on the observations of the RNA density in our structure.

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Jansson, L., Akiyama, B., Ooms, A. et al. Structural basis of template-boundary definition in Tetrahymena telomerase. Nat Struct Mol Biol 22, 883–888 (2015).

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