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Telomerase RNA: A Flexible RNA Scaffold for Telomerase Biosynthesis
Current Biology, Vol. 14, R565–R567, July 27, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.07.013
Dispatch
Telomerase RNA: A Flexible RNA Scaffold for Telomerase Biosynthesis Arthur J. Lustig
Determination of the structure of the yeast telomerase RNA component TLC1 has been hampered by its large size and high rate of evolutionary divergence. But detailed phylogenetic comparisons have now revealed the unusually flexible and modular architecture of this important RNA molecule.
The enzyme telomerase is responsible for addition of Grich simple sequences to the DNA strand that extends from 5′′ to 3′′ at each end of a linear eukaryotic chromosome. Telomerase is unusual in using a template embedded in its RNA component for the addition of simple sequences. Telomerase thereby provides a mechanism to complete replication by compensating for the expected shortening due to loss of the terminal RNA primer after DNA replication. The core telomerase, sufficient for activity in vitro, has two components: telomerase RNA and the telomere reverse transcriptase (TERT). The telomerase holoenzyme in vivo is a far more complex ribonucleoprotein (RNP) particle, with numerous factors that serve essential functions in core telomerase loading, activation and processivity. Included among these proteins are the double-strand break and telomere binding heterodimer Ku, the telomerase recruiter/activator protein Ever-shorter telomere 1 (Est1p), and the Sm antigens that have long been known as a common constituent of RNP particles. The elucidation of telomerase RNA structure is one of the keys to understanding telomerase function and regulation. Phylogenetic comparisons of telomerase RNA sequences have been quite successful in ciliates and vertebrates, and have led to a consensus telomerase RNA structure [1,2]. In particular, RNA domains have been identified that affect template usage [3,4] and TERT association [5]. Significantly, human mutations that alter the equilibrium between different conformational and modification states of telomerase RNAs can result in the disease states dyskeratosis congenita and aplastic anemia [6,7]. Ironically, structural analysis of telomerase RNA in the budding yeast Saccharomyces cerevisiae has lagged behind that in less genetically tractable species, such as ciliates, providing a substantial barrier to mechanistic progress. The yeast telomerase RNA, TLC1, is too large (1.2 kb) and complex for reliable structure derivation based on either first principles or more classical enzymatic techniques. Phylogenetic comparisons have also been limited, given the extraordinarily high rate of divergence of yeast telomerase RNAs, even
Department of Biochemistry, Tulane University Heath Sciences Center, New Orleans, Louisiana 70112, USA. E-mail: [email protected]
when compared to closely related budding yeast species, such as Kluyveromyces [8]. Despite these limitations, some advances have been made in elucidating the domain structure of TLC1 RNA. These include identification of the TERT-binding region in the RNA’s ‘central core’ [5], the stem–bulge required for association with Est1p [9,10] and the stem–loop required for association with yKu70 [11]. But until now these findings could not be integrated in the context of the overall structure of TLC1. This obstacle has now been largely overcome by two groups [12,13] who took advantage of the recently deduced evolutionary relationships among the yeast species that are most closely related to S. cerevisiae [14–16] (Figure 1) by homology and/or synteny — the similarity in the order of the conserved protein-encoding genes that flank TLC1 [13,17]. The species in the Saccharomyces clade I or sensu stricto subgroup — such as S. mikitae, S. paradoxus and S. byanus (Figure 1) — exhibit a level of sequence homology (50–75%) that is ideal for deducing TLC1 RNA structure from the presence of compensatory mutations. In this approach, the RNA sequences from different species are compared and model structures are considered to receive support when pairs of base changes are found that individually would disrupt a unit of secondary structure, but together restore the base pairing that underpins the predicted structure. The TLC1 homologues of the various species have been isolated by parallel construction of plasmid libraries [12,13,18], providing a major tool for structural Saccharomyces cerevisiae 1.0 68 85 69
S. paradoxus S. mikitae
0.78 0.70
S. cariocanus
0.77
S. kudriavzevii
0.66
S. pastorianus
0.59
S. byanus
0.59
67 100
100
64
87
S. castelli
Current Biology
Figure1. Phylogenetic tree of related Saccharomyces species. The tree shows the predicted evolutionary relationships between the species, discussed in the text (not to scale). The upper set of species belong to the clade I (or sensu stricto) group, whereas S. castelli belongs to the more distant clade 3. The number above or below each branch refers to the bootstrapping value, a statistical reflection of the degree of relatedness and homology. The break lines represent a separation of larger degrees of divergence. The numbers on the right refer to the fractional homology of the TLC1 gene relative to Saccharomyces cerevisiae. (Adapted from [16].)
Dispatch R566
Table 1. A preliminary outline of TLC1 RNA structure–function relationships. Associations
Stem/loop structures
Functions
Vertebrate correlation
Central core
Est2p binding
III, V, VI; III/VI pseudoknot
Template boundary placement; processivity
Pseudoknot functionally conserved
EST 1 ARM
Est1p association
IVA, IVB, IVC bulge
Telomerase loading +/– activation
?
KU ARM
yKu70 binding
IIA, IIB, IIC, IID, IID-1
Healing and telomerase efficiency; ? end protection
Terminal arm
Sm antigen binding
Stem 1, VIIA, VIIB, VIIC
RNP nucleation; end processing
?
See [12,13,18] and references therein for the data on which this table is based. Question marks refer to an unknown relationship between yeasts and vertebrates.
and functional analyses of rapidly diverging RNAs and proteins. The Wellinger [12] and Cech [13] laboratories have now used this approach to come up with a working model for TLC1 RNA structure (Table 1). The major elements of the two structures are strikingly similar, consisting of a central ‘hub’ with three major extended arms. At a molecular level, a central core domain containing a pseudo-knot (or comparable structure) adjacent to the template sequence [5] is conserved in most eukaryotes, and in S. cerevisiae this is required for association of the TERT component Est2p [6,8,19]. The three arms that emanate from the core region associate with proteins that have distinct functions. The ‘terminal arm’ contains binding sites for Sm antigens near the TLC1 3′′ and 5′′ ends. The ‘Est1’ arm contains multiple stem–loop structures including the stem-bulged region essential for RNA’s association with Est1p. The ‘Ku’ arm includes the stem–loop structure previously identified as the site of yKu70 association. The model is consistent with the patterns of site cleavage by RNaseH, which cleaves only single-stranded RNA, providing partial biochemical confirmation of the phylogenetic predictions. The Est2p association site within the core domain has been characterized further through the effect of single and compensatory mutations by a phylogenetic study that used the more distant yeasts S. kluyverii and Kluyveromyces lactis [18]. This analysis indicates that Est2p associates with a stem–loop region coupled with additional sequences that may form a pseudo-knot, one of the few highly conserved elements within all telomerase RNAs, or another structure of as yet undetermined character. Nonetheless, the replacement of the Est2p association site within the S. cerevisiae TLC1 by either the ciliate or human telomerase RNA pseudoknot restores Est2p binding and telomerase function, an indication of both the evolutionary conservation and structural flexibility of telomerase RNA. Zappulla and Cech [13] took this analysis one step further by determining the sequences in TLC1 RNA required for association with Est1p. Est1p — in conjunction with other proteins, including the central telomere single-stranded DNA binding protein Cdc13p [20] — ensures telomerase recruitment and activation within the appropriate cell-cycle window. The stem–bulge secondary structure in TLC1 RNA, as defined by previous studies in Cech’s lab, is not only essential [9], but also sufficient for the in vitro association of Est1p with TLC1 RNA. Remarkably, the authors [13] found that
transfer of this secondary structure ‘module’ to other regions of TLC1 permits Est1p binding in vitro and full telomerase activity in vivo. The primary functional determinant in TLC1 RNA thus appears to be at the secondary structure level. This finding suggests that the TLC1 RNA structure consists of a scaffold into which are plugged modular binding sites for telomeric regulatory proteins. Supporting this view, the Sm antigen sites do not have to be placed in a specific region of TLC1 relative to the RNA ends [13]. The modular secondary structure of TLC1 RNA is unusual and, given the need of the corresponding binding factors to interact in vivo, is likely to also be accompanied by significant flexibility in the RNA’s tertiary structure. Vertebrate and ciliate telomerase RNAs seem to lack the flexibility and modularity of the multifunctional TLC1 RNA. The expected rate of divergence of RNA is difficult to calculate, given its dependence on the functional conservation of specific residues. A high rate of yeast TLC1 sequence divergence, however, seems to mirror the high degree of variation among functionally homologous telomere-binding proteins of S. cerevisiae and its more distant relatives. It is possible that additional requirements for telomerase RNA in some organisms are incompatible with the yeast flexible scaffold; but perhaps more likely is the possibility that the flexible scaffold structure confers an as yet unknown selective advantage in budding yeasts. The synergy between molecular biology and phylogenetic studies of TLC1 bodes well for future successes that will address key issues that should ultimately reveal the secrets underlying the mechanism of telomerase activity. References 1. Romero, D.P., and Blackburn, E.H. (1991). A conserved secondary structure for telomerase RNA. Cell 67, 343-353. 2. Chen, J.L., Blasco, M.A., and Greider, C.W. (2000). Secondary structure of vertebrate telomerase RNA. Cell 100, 503-514. 3. Chen, J.L., and Greider, C.W. (2003). Template boundary definition in mammalian telomerase. Genes Dev. 17, 2747-2752. 4. Lai, C.K., Miller, M.C., and Collins, K. (2003). Roles for RNA in telomerase nucleotide and repeat addition processivity. Mol. Cell 11, 1673-1683. 5. Ly, H., Blackburn, E.H., and Parslow, T.G. (2003). Comprehensive structure-function analysis of the core domain of human telomerase RNA. Mol. Cell Biol. 23, 6849-6856. 6. Comolli, L.R., Smirnov, I., Xu, L., Blackburn, E.H., and James, T.L. (2002). A molecular switch underlies a human telomerase disease. Proc. Natl. Acad. Sci. USA 99, 16998-17003. 7. Chen, J.L., and Greider, C.W. (2004). Telomerase RNA structure and function: implications for dyskeratosis congenita. Trends Biochem. Sci. 29, 183-192.
Current Biology R567
8. Tzfati, Y., Knight, Z., Roy, J., and Blackburn, E.H. (2003). A novel pseudoknot element is essential for the action of a yeast telomerase. Genes Dev. 17, 1779-1788. 9. Seto, A.G., Livengood, A.J., Tzfati, Y., Blackburn, E.H., and Cech, T.R. (2002). A bulged stem tethers Est1p to telomerase RNA in budding yeast. Genes Dev. 16, 2800-2812. 10. Livengood, A.J., Zaug, A.J., and Cech, T.R. (2002). Essential regions of Saccharomyces cerevisiae telomerase RNA: separate elements for Est1p and Est2p interaction. Mol. Cell Biol. 22, 2366-2374. 11. Stellwagen, A.E., Haimberger, Z.W., Veatch, J.R., and Gottschling, D.E. (2003). Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev. 17, 2384-2395. 12. Dandjinou, A.T., Lévesque, N., Larose, S., Lucier, J.-F., Elela, S.A., and Wellinger, R. (2004). A phylogenetically based secondary structure for the yeast telomerase RNA. Curr. Biol. July 13 issue. 13. Zappulla, D.C., and Cech, T.R. (2004). Yeast telomerase RNA: a flexible scaffold for protein subunits. Proc. Natl. Acad. Sci. USA, in press. 14. Kellis, M., Patterson, N., Endrizzi, M., Birren, B., and Lander, E.S. (2003). Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature 423, 241-254. 15. Cliften, P., Sudarsanam, P., Desikan, A., Fulton, L., Fulton, B., Majors, J., Waterston, R., Cohen, B.A., and Johnston, M. (2003). Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science 301, 71-76. 16. Kurtzman, C.P., and Robnett, C.J. (2003). Phylogenetic relationships among yeasts of the ‘Saccharomyces complex’ determined from multigene sequence analyses. FEMS Yeast Res 3, 417-432. 17. Dietrich, F.S., Voegeli, S., Brachat, S., Lerch, A., Gates, K., Steiner, S., Mohr, C., Pohlmann, R., Luedi, P., Choi, S., Wing, R.A., Flavier, A., Gaffney, T.D., and Philippsen, P. (2004). The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome. Science 304, 304-307. 18. Chappell, A.S., and Lundblad, V. (2004). Structural elements required for association of the Saccharomyces cerevisiae telomerase RNA with the Est2 reverse transcriptase. Mol. Cell Biol., in press. 19. Martin-Rivera, L., and Blasco, M.A. (2001). Identification of functional domains and dominant negative mutations in vertebrate telomerase RNA using an in vivo reconstitution system. J. Biol. Chem. 276, 5856-5865. 20. Evans, S.K., and Lundblad, V. (2002). The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance. Genetics 162, 1101-1115.
Dispatch
Telomerase RNA: A Flexible RNA Scaffold for Telomerase Biosynthesis Arthur J. Lustig
Determination of the structure of the yeast telomerase RNA component TLC1 has been hampered by its large size and high rate of evolutionary divergence. But detailed phylogenetic comparisons have now revealed the unusually flexible and modular architecture of this important RNA molecule.
The enzyme telomerase is responsible for addition of Grich simple sequences to the DNA strand that extends from 5′′ to 3′′ at each end of a linear eukaryotic chromosome. Telomerase is unusual in using a template embedded in its RNA component for the addition of simple sequences. Telomerase thereby provides a mechanism to complete replication by compensating for the expected shortening due to loss of the terminal RNA primer after DNA replication. The core telomerase, sufficient for activity in vitro, has two components: telomerase RNA and the telomere reverse transcriptase (TERT). The telomerase holoenzyme in vivo is a far more complex ribonucleoprotein (RNP) particle, with numerous factors that serve essential functions in core telomerase loading, activation and processivity. Included among these proteins are the double-strand break and telomere binding heterodimer Ku, the telomerase recruiter/activator protein Ever-shorter telomere 1 (Est1p), and the Sm antigens that have long been known as a common constituent of RNP particles. The elucidation of telomerase RNA structure is one of the keys to understanding telomerase function and regulation. Phylogenetic comparisons of telomerase RNA sequences have been quite successful in ciliates and vertebrates, and have led to a consensus telomerase RNA structure [1,2]. In particular, RNA domains have been identified that affect template usage [3,4] and TERT association [5]. Significantly, human mutations that alter the equilibrium between different conformational and modification states of telomerase RNAs can result in the disease states dyskeratosis congenita and aplastic anemia [6,7]. Ironically, structural analysis of telomerase RNA in the budding yeast Saccharomyces cerevisiae has lagged behind that in less genetically tractable species, such as ciliates, providing a substantial barrier to mechanistic progress. The yeast telomerase RNA, TLC1, is too large (1.2 kb) and complex for reliable structure derivation based on either first principles or more classical enzymatic techniques. Phylogenetic comparisons have also been limited, given the extraordinarily high rate of divergence of yeast telomerase RNAs, even
Department of Biochemistry, Tulane University Heath Sciences Center, New Orleans, Louisiana 70112, USA. E-mail: [email protected]
when compared to closely related budding yeast species, such as Kluyveromyces [8]. Despite these limitations, some advances have been made in elucidating the domain structure of TLC1 RNA. These include identification of the TERT-binding region in the RNA’s ‘central core’ [5], the stem–bulge required for association with Est1p [9,10] and the stem–loop required for association with yKu70 [11]. But until now these findings could not be integrated in the context of the overall structure of TLC1. This obstacle has now been largely overcome by two groups [12,13] who took advantage of the recently deduced evolutionary relationships among the yeast species that are most closely related to S. cerevisiae [14–16] (Figure 1) by homology and/or synteny — the similarity in the order of the conserved protein-encoding genes that flank TLC1 [13,17]. The species in the Saccharomyces clade I or sensu stricto subgroup — such as S. mikitae, S. paradoxus and S. byanus (Figure 1) — exhibit a level of sequence homology (50–75%) that is ideal for deducing TLC1 RNA structure from the presence of compensatory mutations. In this approach, the RNA sequences from different species are compared and model structures are considered to receive support when pairs of base changes are found that individually would disrupt a unit of secondary structure, but together restore the base pairing that underpins the predicted structure. The TLC1 homologues of the various species have been isolated by parallel construction of plasmid libraries [12,13,18], providing a major tool for structural Saccharomyces cerevisiae 1.0 68 85 69
S. paradoxus S. mikitae
0.78 0.70
S. cariocanus
0.77
S. kudriavzevii
0.66
S. pastorianus
0.59
S. byanus
0.59
67 100
100
64
87
S. castelli
Current Biology
Figure1. Phylogenetic tree of related Saccharomyces species. The tree shows the predicted evolutionary relationships between the species, discussed in the text (not to scale). The upper set of species belong to the clade I (or sensu stricto) group, whereas S. castelli belongs to the more distant clade 3. The number above or below each branch refers to the bootstrapping value, a statistical reflection of the degree of relatedness and homology. The break lines represent a separation of larger degrees of divergence. The numbers on the right refer to the fractional homology of the TLC1 gene relative to Saccharomyces cerevisiae. (Adapted from [16].)
Dispatch R566
Table 1. A preliminary outline of TLC1 RNA structure–function relationships. Associations
Stem/loop structures
Functions
Vertebrate correlation
Central core
Est2p binding
III, V, VI; III/VI pseudoknot
Template boundary placement; processivity
Pseudoknot functionally conserved
EST 1 ARM
Est1p association
IVA, IVB, IVC bulge
Telomerase loading +/– activation
?
KU ARM
yKu70 binding
IIA, IIB, IIC, IID, IID-1
Healing and telomerase efficiency; ? end protection
Terminal arm
Sm antigen binding
Stem 1, VIIA, VIIB, VIIC
RNP nucleation; end processing
?
See [12,13,18] and references therein for the data on which this table is based. Question marks refer to an unknown relationship between yeasts and vertebrates.
and functional analyses of rapidly diverging RNAs and proteins. The Wellinger [12] and Cech [13] laboratories have now used this approach to come up with a working model for TLC1 RNA structure (Table 1). The major elements of the two structures are strikingly similar, consisting of a central ‘hub’ with three major extended arms. At a molecular level, a central core domain containing a pseudo-knot (or comparable structure) adjacent to the template sequence [5] is conserved in most eukaryotes, and in S. cerevisiae this is required for association of the TERT component Est2p [6,8,19]. The three arms that emanate from the core region associate with proteins that have distinct functions. The ‘terminal arm’ contains binding sites for Sm antigens near the TLC1 3′′ and 5′′ ends. The ‘Est1’ arm contains multiple stem–loop structures including the stem-bulged region essential for RNA’s association with Est1p. The ‘Ku’ arm includes the stem–loop structure previously identified as the site of yKu70 association. The model is consistent with the patterns of site cleavage by RNaseH, which cleaves only single-stranded RNA, providing partial biochemical confirmation of the phylogenetic predictions. The Est2p association site within the core domain has been characterized further through the effect of single and compensatory mutations by a phylogenetic study that used the more distant yeasts S. kluyverii and Kluyveromyces lactis [18]. This analysis indicates that Est2p associates with a stem–loop region coupled with additional sequences that may form a pseudo-knot, one of the few highly conserved elements within all telomerase RNAs, or another structure of as yet undetermined character. Nonetheless, the replacement of the Est2p association site within the S. cerevisiae TLC1 by either the ciliate or human telomerase RNA pseudoknot restores Est2p binding and telomerase function, an indication of both the evolutionary conservation and structural flexibility of telomerase RNA. Zappulla and Cech [13] took this analysis one step further by determining the sequences in TLC1 RNA required for association with Est1p. Est1p — in conjunction with other proteins, including the central telomere single-stranded DNA binding protein Cdc13p [20] — ensures telomerase recruitment and activation within the appropriate cell-cycle window. The stem–bulge secondary structure in TLC1 RNA, as defined by previous studies in Cech’s lab, is not only essential [9], but also sufficient for the in vitro association of Est1p with TLC1 RNA. Remarkably, the authors [13] found that
transfer of this secondary structure ‘module’ to other regions of TLC1 permits Est1p binding in vitro and full telomerase activity in vivo. The primary functional determinant in TLC1 RNA thus appears to be at the secondary structure level. This finding suggests that the TLC1 RNA structure consists of a scaffold into which are plugged modular binding sites for telomeric regulatory proteins. Supporting this view, the Sm antigen sites do not have to be placed in a specific region of TLC1 relative to the RNA ends [13]. The modular secondary structure of TLC1 RNA is unusual and, given the need of the corresponding binding factors to interact in vivo, is likely to also be accompanied by significant flexibility in the RNA’s tertiary structure. Vertebrate and ciliate telomerase RNAs seem to lack the flexibility and modularity of the multifunctional TLC1 RNA. The expected rate of divergence of RNA is difficult to calculate, given its dependence on the functional conservation of specific residues. A high rate of yeast TLC1 sequence divergence, however, seems to mirror the high degree of variation among functionally homologous telomere-binding proteins of S. cerevisiae and its more distant relatives. It is possible that additional requirements for telomerase RNA in some organisms are incompatible with the yeast flexible scaffold; but perhaps more likely is the possibility that the flexible scaffold structure confers an as yet unknown selective advantage in budding yeasts. The synergy between molecular biology and phylogenetic studies of TLC1 bodes well for future successes that will address key issues that should ultimately reveal the secrets underlying the mechanism of telomerase activity. References 1. Romero, D.P., and Blackburn, E.H. (1991). A conserved secondary structure for telomerase RNA. Cell 67, 343-353. 2. Chen, J.L., Blasco, M.A., and Greider, C.W. (2000). Secondary structure of vertebrate telomerase RNA. Cell 100, 503-514. 3. Chen, J.L., and Greider, C.W. (2003). Template boundary definition in mammalian telomerase. Genes Dev. 17, 2747-2752. 4. Lai, C.K., Miller, M.C., and Collins, K. (2003). Roles for RNA in telomerase nucleotide and repeat addition processivity. Mol. Cell 11, 1673-1683. 5. Ly, H., Blackburn, E.H., and Parslow, T.G. (2003). Comprehensive structure-function analysis of the core domain of human telomerase RNA. Mol. Cell Biol. 23, 6849-6856. 6. Comolli, L.R., Smirnov, I., Xu, L., Blackburn, E.H., and James, T.L. (2002). A molecular switch underlies a human telomerase disease. Proc. Natl. Acad. Sci. USA 99, 16998-17003. 7. Chen, J.L., and Greider, C.W. (2004). Telomerase RNA structure and function: implications for dyskeratosis congenita. Trends Biochem. Sci. 29, 183-192.
Current Biology R567
8. Tzfati, Y., Knight, Z., Roy, J., and Blackburn, E.H. (2003). A novel pseudoknot element is essential for the action of a yeast telomerase. Genes Dev. 17, 1779-1788. 9. Seto, A.G., Livengood, A.J., Tzfati, Y., Blackburn, E.H., and Cech, T.R. (2002). A bulged stem tethers Est1p to telomerase RNA in budding yeast. Genes Dev. 16, 2800-2812. 10. Livengood, A.J., Zaug, A.J., and Cech, T.R. (2002). Essential regions of Saccharomyces cerevisiae telomerase RNA: separate elements for Est1p and Est2p interaction. Mol. Cell Biol. 22, 2366-2374. 11. Stellwagen, A.E., Haimberger, Z.W., Veatch, J.R., and Gottschling, D.E. (2003). Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev. 17, 2384-2395. 12. Dandjinou, A.T., Lévesque, N., Larose, S., Lucier, J.-F., Elela, S.A., and Wellinger, R. (2004). A phylogenetically based secondary structure for the yeast telomerase RNA. Curr. Biol. July 13 issue. 13. Zappulla, D.C., and Cech, T.R. (2004). Yeast telomerase RNA: a flexible scaffold for protein subunits. Proc. Natl. Acad. Sci. USA, in press. 14. Kellis, M., Patterson, N., Endrizzi, M., Birren, B., and Lander, E.S. (2003). Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature 423, 241-254. 15. Cliften, P., Sudarsanam, P., Desikan, A., Fulton, L., Fulton, B., Majors, J., Waterston, R., Cohen, B.A., and Johnston, M. (2003). Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science 301, 71-76. 16. Kurtzman, C.P., and Robnett, C.J. (2003). Phylogenetic relationships among yeasts of the ‘Saccharomyces complex’ determined from multigene sequence analyses. FEMS Yeast Res 3, 417-432. 17. Dietrich, F.S., Voegeli, S., Brachat, S., Lerch, A., Gates, K., Steiner, S., Mohr, C., Pohlmann, R., Luedi, P., Choi, S., Wing, R.A., Flavier, A., Gaffney, T.D., and Philippsen, P. (2004). The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome. Science 304, 304-307. 18. Chappell, A.S., and Lundblad, V. (2004). Structural elements required for association of the Saccharomyces cerevisiae telomerase RNA with the Est2 reverse transcriptase. Mol. Cell Biol., in press. 19. Martin-Rivera, L., and Blasco, M.A. (2001). Identification of functional domains and dominant negative mutations in vertebrate telomerase RNA using an in vivo reconstitution system. J. Biol. Chem. 276, 5856-5865. 20. Evans, S.K., and Lundblad, V. (2002). The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance. Genetics 162, 1101-1115.