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. 1999 Jun 8;96(12):6621-5.
doi: 10.1073/pnas.96.12.6621.

The telomerase RNA pseudoknot is critical for the stable assembly of a catalytically active ribonucleoprotein

D Gilley  1 , E H Blackburn
Affiliations

The telomerase RNA pseudoknot is critical for the stable assembly of a catalytically active ribonucleoprotein

D Gilley et al. Proc Natl Acad Sci U S A. .

Abstract

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes telomeric DNA. A pseudoknot structure is phylogenetically conserved within the RNA component of telomerase in all ciliated protozoans examined. Here, we report that disruptions of the pseudoknot base pairing within the telomerase RNA from Tetrahymena thermophila prevent the stable assembly in vivo of an active telomerase. Restoring the base-pairing potential of the pseudoknot by compensatory changes restores telomerase activity to essentially wild-type levels. Therefore, the pseudoknot topology rather than sequence is critical for an active telomerase. Furthermore, we show that disruption of the pseudoknot prevents the association of the RNA with the reverse transcriptase protein subunit of telomerase. Thus, we provide an example of a structural motif within the telomerase RNA that is required for telomerase function and identify the domain that is required for telomerase complex formation. Hence, we identify a biological role for a pseudoknot: promoting the stable assembly of a catalytically active ribonucleoprotein.

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Figures

Figure 1
Figure 1
Ciliate TER structure and pseudoknot sequence alterations. (A) The conserved core structure of ciliate TER determined by phylogenetic covariation and structural probing (4), highlighting the pseudoknot domain that forms into a quasicontinuous helix on stacking of the two stems, and the wild-type template sequence from T. thermophila. (B) Detail of the pseudoknot domain from T. thermophila with sequence, position numbers, and standard designations of stems 1 and 2 as well as loops 1 and 2. (C) Sequence alterations used within the pseudoknot domain. The top line shows the wild-type pseudoknot RNA sequence from T. thermophila. Specific changes are shown directly below the wild-type residues that have been changed. Comp 1 and Comp 2, compensatory mutations 1 and 2.
Figure 2
Figure 2
Relative telomerase activity levels of disruptions and compensatory alterations within the pseudoknot domain compared with wild-type telomerase. Telomerase activity levels were normalized by using cell numbers and total protein content of partially purified preparations. The results represent an average of three separate experiments with a minimum of two lines for each sample. (A) Two-base pseudoknot disruptions, 71–72G and 84–85C, and the corresponding compensatory alteration, Comp 1, compared with activity levels for wild-type telomerase (WT). (B) Four-base pseudoknot disruption, 84–87C, and the corresponding compensatory alteration, Comp 2, compared with activity level for wild-type telomerase (WT).
Figure 3
Figure 3
Marking the template in combination with pseudoknot alterations. (A) Standard telomerase in vitro assay reactions separated on 10% PAGE. Cells were transformed with various pseudoknot wild-type or pseudoknot-altered TER genes that were marked at the template with the 43A template mutation. Lane 1, wild-type (WT) at pseudoknot; lane 2, Comp 1; lane 3, Comp 2; lane 4, 84–87C. (B) Schematic of 43A and wild-type templates. Telomerase with a 43A template change synthesizes a seven-base repeat consisting of G4T3. Wild-type telomerase synthesizes a six-base repeat consisting of G4T2. Note that cell numbers and protein content were not normalized between these different lines and that telomerase was harvested from cells in stationary phase culture.
Figure 4
Figure 4
TER-associated protein complexes separated by native gel electrophoresis. (A) Northern analysis of native complexes. Northern blots probed with either a wild-type-specific end-labeled oligonucleotide (5′-ATTTGAACCTAATTGG-3′) that annealed only to wild-type TER (lanes 1–6) or a mutant-specific end-labeled oligonucleotide (5′-ATTTGGGGGTAATTGG-3′) that annealed only to 84–87C and Comp 2 TERs (lanes 7–12). These antisense oligonucleotides anneal to residues 92–77 of T. thermophila TER. Lane 1 and 7, wild-type; lane 2 and 8, 84–87C; lane 3 and 9, Comp 2; lane 4 and 10, wild-type with 43A template; lane 5 and 11, Comp 2 with 43A template; lane 6 and 12, 84–87C with 43A template. (B) Western analysis of native complexes. Western blots probed with antibody against p95 (lanes 13–18), p80 (lanes 19–24), or p133 (lanes 25–30). Lanes 13, 19, and 25, wild-type; lanes 14, 20, and 26, Comp 2; lanes 15, 21, and 27, 84–87C; lanes 16, 22, and 28, wild-type with 43A template; lanes 17, 23, and 29, Comp 2 with 43A template; lanes 18, 24, and 30, 84–87C.
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