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Triple-helix structure in telomerase RNA contributes to catalysis.

Qiao F, Cech TR - Nat. Struct. Mol. Biol. (2008)

Bottom Line: Its intrinsic RNA subunit provides the template for synthesis of telomeric DNA by the reverse-transcriptase (TERT) subunit and tethers other proteins into the ribonucleoprotein (RNP) complex.We report that a phylogenetically conserved triple helix within a pseudoknot structure of this RNA contributes to telomerase activity but not by binding the TERT protein.The role of RNA in telomerase catalysis may have been acquired relatively recently or, alternatively, telomerase may be a molecular fossil representing an evolutionary link between RNA enzymes and RNP enzymes.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, Colorado 80309-0215, USA.

ABSTRACT
Telomerase is responsible for replication of the ends of linear chromosomes in most eukaryotes. Its intrinsic RNA subunit provides the template for synthesis of telomeric DNA by the reverse-transcriptase (TERT) subunit and tethers other proteins into the ribonucleoprotein (RNP) complex. We report that a phylogenetically conserved triple helix within a pseudoknot structure of this RNA contributes to telomerase activity but not by binding the TERT protein. Instead, 2'-OH groups protruding from the triple helix participate in both yeast and human telomerase catalysis; they may orient the primer-template relative to the active site in a manner analogous to group I ribozymes. The role of RNA in telomerase catalysis may have been acquired relatively recently or, alternatively, telomerase may be a molecular fossil representing an evolutionary link between RNA enzymes and RNP enzymes.

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The pseudoknot region of the S. cerevisiae telomerase RNA contains a triple-helix structure. (a) The sequences and the secondary structures of the triple-helix and adjacent regions in the context of the Micro-T 170 telomerase RNA (see Supplementary Fig. 3 for detail). The three dotted lines indicate the base triples best supported by this work; an additional flanking U-A•U triple may also form but was not tested here. (b) Base triples formed by U-A•U, C-G•C and C-G•U, respectively. All involve formation of Hoogsteen base pairs in the major groove of the Watson-Crick duplex. In the case of the C-G•C triple, protonation of N3 of cytosine at pH below its pKa (free nucleotide has pKa ~4.2) could provide an additional hydrogen bond (gray). (c) In vitro telomerase activities of the telomerase RNA triple helix–disrupting mutants 11c_3G (A804–A806→GGG), 6a_3C (U757–A759→CCC), 4b_3C (U746–U748→CCC) and the triple helix–restoring mutants 3GCC-1 (A804–A806→GGG/U757–A759→CCC/U746–U748→CCC), 3GCC-2 (A804–A806→GGG/U757–A759→CCC/U745–U747→CCC) and 3GCC-3 (A804–A806→GGG/U757–A759→CCC/U744–U746→CCC). The activities of individual mutants were normalized to the wild-type (WT) activity. (d) Telomere-length analysis of the telomerase RNA triple helix–disrupting mutants and triple helix–restoring mutants compared with the wild-type TLC1. Southern blot shows lengths of telomeric XhoI restriction fragments from cells harboring different TLC1 constructs and grown for 125 generations. Duplicate lanes represent two independent clones for each mutant. A probe that hybridizes to both strands of telomeric DNA was used. The markers shown were radiolabeled with 2-Log DNA Ladder (NEB). Y′ telomeres are preceded by a repeated DNA sequence that contains an XhoI restriction site.
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Figure 2: The pseudoknot region of the S. cerevisiae telomerase RNA contains a triple-helix structure. (a) The sequences and the secondary structures of the triple-helix and adjacent regions in the context of the Micro-T 170 telomerase RNA (see Supplementary Fig. 3 for detail). The three dotted lines indicate the base triples best supported by this work; an additional flanking U-A•U triple may also form but was not tested here. (b) Base triples formed by U-A•U, C-G•C and C-G•U, respectively. All involve formation of Hoogsteen base pairs in the major groove of the Watson-Crick duplex. In the case of the C-G•C triple, protonation of N3 of cytosine at pH below its pKa (free nucleotide has pKa ~4.2) could provide an additional hydrogen bond (gray). (c) In vitro telomerase activities of the telomerase RNA triple helix–disrupting mutants 11c_3G (A804–A806→GGG), 6a_3C (U757–A759→CCC), 4b_3C (U746–U748→CCC) and the triple helix–restoring mutants 3GCC-1 (A804–A806→GGG/U757–A759→CCC/U746–U748→CCC), 3GCC-2 (A804–A806→GGG/U757–A759→CCC/U745–U747→CCC) and 3GCC-3 (A804–A806→GGG/U757–A759→CCC/U744–U746→CCC). The activities of individual mutants were normalized to the wild-type (WT) activity. (d) Telomere-length analysis of the telomerase RNA triple helix–disrupting mutants and triple helix–restoring mutants compared with the wild-type TLC1. Southern blot shows lengths of telomeric XhoI restriction fragments from cells harboring different TLC1 constructs and grown for 125 generations. Duplicate lanes represent two independent clones for each mutant. A probe that hybridizes to both strands of telomeric DNA was used. The markers shown were radiolabeled with 2-Log DNA Ladder (NEB). Y′ telomeres are preceded by a repeated DNA sequence that contains an XhoI restriction site.

Mentions: The predicted U-A•U base triples were tested by mutating them to C-G•C, which is the best structural mimic of U-A•U; protonation of the Hoogsteen-paired cytosine would provide an additional hydrogen bond (Fig. 2a,b). None of the mutants with mutations in one of the three individual strands (AAA mutated to GGG, and UUU mutated to CCC) had more than 10% of the wild-type telomerase activity (Fig. 2c). Previous efforts to restore the Stem 2 Watson-Crick base pairs by swapping the sequences of the two strands failed to rescue telomere shortening19, indicating that duplex formation is not sufficient for telomerase activity. Notably, when we combined all three sets of mutations, telomerase activity was rescued to the wild-type level (Fig. 2c, right). This result strongly indicates the existence of a triple helix in the S. cerevisiae telomerase RNA core region. As two of the three block mutants (3GCC-1 and 3GCC-2) showed recovery of activity, there seems to be some flexibility in which portion of the third strand forms the Hoogsteen base pairs. Furthermore, a mutant with C-G•U substituting for U-A•U, in which only one Hoogsteen hydrogen bond could be formed (Fig. 2b), also provided more than 60% of the wild-type telomerase activity (data not shown); thus, protonation of N3 of cytosine may be optional in the case of these C-G•C triples, because the entropic cost of forming the triple helix is ameliorated by the existence of the adjacent pseudoknot.


Triple-helix structure in telomerase RNA contributes to catalysis.

Qiao F, Cech TR - Nat. Struct. Mol. Biol. (2008)

The pseudoknot region of the S. cerevisiae telomerase RNA contains a triple-helix structure. (a) The sequences and the secondary structures of the triple-helix and adjacent regions in the context of the Micro-T 170 telomerase RNA (see Supplementary Fig. 3 for detail). The three dotted lines indicate the base triples best supported by this work; an additional flanking U-A•U triple may also form but was not tested here. (b) Base triples formed by U-A•U, C-G•C and C-G•U, respectively. All involve formation of Hoogsteen base pairs in the major groove of the Watson-Crick duplex. In the case of the C-G•C triple, protonation of N3 of cytosine at pH below its pKa (free nucleotide has pKa ~4.2) could provide an additional hydrogen bond (gray). (c) In vitro telomerase activities of the telomerase RNA triple helix–disrupting mutants 11c_3G (A804–A806→GGG), 6a_3C (U757–A759→CCC), 4b_3C (U746–U748→CCC) and the triple helix–restoring mutants 3GCC-1 (A804–A806→GGG/U757–A759→CCC/U746–U748→CCC), 3GCC-2 (A804–A806→GGG/U757–A759→CCC/U745–U747→CCC) and 3GCC-3 (A804–A806→GGG/U757–A759→CCC/U744–U746→CCC). The activities of individual mutants were normalized to the wild-type (WT) activity. (d) Telomere-length analysis of the telomerase RNA triple helix–disrupting mutants and triple helix–restoring mutants compared with the wild-type TLC1. Southern blot shows lengths of telomeric XhoI restriction fragments from cells harboring different TLC1 constructs and grown for 125 generations. Duplicate lanes represent two independent clones for each mutant. A probe that hybridizes to both strands of telomeric DNA was used. The markers shown were radiolabeled with 2-Log DNA Ladder (NEB). Y′ telomeres are preceded by a repeated DNA sequence that contains an XhoI restriction site.
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Figure 2: The pseudoknot region of the S. cerevisiae telomerase RNA contains a triple-helix structure. (a) The sequences and the secondary structures of the triple-helix and adjacent regions in the context of the Micro-T 170 telomerase RNA (see Supplementary Fig. 3 for detail). The three dotted lines indicate the base triples best supported by this work; an additional flanking U-A•U triple may also form but was not tested here. (b) Base triples formed by U-A•U, C-G•C and C-G•U, respectively. All involve formation of Hoogsteen base pairs in the major groove of the Watson-Crick duplex. In the case of the C-G•C triple, protonation of N3 of cytosine at pH below its pKa (free nucleotide has pKa ~4.2) could provide an additional hydrogen bond (gray). (c) In vitro telomerase activities of the telomerase RNA triple helix–disrupting mutants 11c_3G (A804–A806→GGG), 6a_3C (U757–A759→CCC), 4b_3C (U746–U748→CCC) and the triple helix–restoring mutants 3GCC-1 (A804–A806→GGG/U757–A759→CCC/U746–U748→CCC), 3GCC-2 (A804–A806→GGG/U757–A759→CCC/U745–U747→CCC) and 3GCC-3 (A804–A806→GGG/U757–A759→CCC/U744–U746→CCC). The activities of individual mutants were normalized to the wild-type (WT) activity. (d) Telomere-length analysis of the telomerase RNA triple helix–disrupting mutants and triple helix–restoring mutants compared with the wild-type TLC1. Southern blot shows lengths of telomeric XhoI restriction fragments from cells harboring different TLC1 constructs and grown for 125 generations. Duplicate lanes represent two independent clones for each mutant. A probe that hybridizes to both strands of telomeric DNA was used. The markers shown were radiolabeled with 2-Log DNA Ladder (NEB). Y′ telomeres are preceded by a repeated DNA sequence that contains an XhoI restriction site.
Mentions: The predicted U-A•U base triples were tested by mutating them to C-G•C, which is the best structural mimic of U-A•U; protonation of the Hoogsteen-paired cytosine would provide an additional hydrogen bond (Fig. 2a,b). None of the mutants with mutations in one of the three individual strands (AAA mutated to GGG, and UUU mutated to CCC) had more than 10% of the wild-type telomerase activity (Fig. 2c). Previous efforts to restore the Stem 2 Watson-Crick base pairs by swapping the sequences of the two strands failed to rescue telomere shortening19, indicating that duplex formation is not sufficient for telomerase activity. Notably, when we combined all three sets of mutations, telomerase activity was rescued to the wild-type level (Fig. 2c, right). This result strongly indicates the existence of a triple helix in the S. cerevisiae telomerase RNA core region. As two of the three block mutants (3GCC-1 and 3GCC-2) showed recovery of activity, there seems to be some flexibility in which portion of the third strand forms the Hoogsteen base pairs. Furthermore, a mutant with C-G•U substituting for U-A•U, in which only one Hoogsteen hydrogen bond could be formed (Fig. 2b), also provided more than 60% of the wild-type telomerase activity (data not shown); thus, protonation of N3 of cytosine may be optional in the case of these C-G•C triples, because the entropic cost of forming the triple helix is ameliorated by the existence of the adjacent pseudoknot.

Bottom Line: Its intrinsic RNA subunit provides the template for synthesis of telomeric DNA by the reverse-transcriptase (TERT) subunit and tethers other proteins into the ribonucleoprotein (RNP) complex.We report that a phylogenetically conserved triple helix within a pseudoknot structure of this RNA contributes to telomerase activity but not by binding the TERT protein.The role of RNA in telomerase catalysis may have been acquired relatively recently or, alternatively, telomerase may be a molecular fossil representing an evolutionary link between RNA enzymes and RNP enzymes.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, Colorado 80309-0215, USA.

ABSTRACT
Telomerase is responsible for replication of the ends of linear chromosomes in most eukaryotes. Its intrinsic RNA subunit provides the template for synthesis of telomeric DNA by the reverse-transcriptase (TERT) subunit and tethers other proteins into the ribonucleoprotein (RNP) complex. We report that a phylogenetically conserved triple helix within a pseudoknot structure of this RNA contributes to telomerase activity but not by binding the TERT protein. Instead, 2'-OH groups protruding from the triple helix participate in both yeast and human telomerase catalysis; they may orient the primer-template relative to the active site in a manner analogous to group I ribozymes. The role of RNA in telomerase catalysis may have been acquired relatively recently or, alternatively, telomerase may be a molecular fossil representing an evolutionary link between RNA enzymes and RNP enzymes.

Show MeSH
Related in: MedlinePlus