A conserved secondary structure for telomerase RNA

Cell, Vol. 67, 343-353, October 18, 1991,Copyright© 1991 by Cell Press

A Conserved Secondary Structure for Telomerase RNA Daniel P. Romero and Elizabeth H. Blackburn Department of Microbiology and Immunology University of California, San Francisco San Francisco, California 94143

Summary The RNA moiety of the ribonucleoprotein enzyme telomerase contains the template for telomeric DNA synthesis. We present a secondary structure model for telomerase RNA, derived by a phylogenetic comparative analysis of telomerase RNAs from seven tetrahymenine ciliates. The telomerase RNA genes from Tetrahymena malaccensis, T. pyriformis, T. hyperangularis, T. pigmentosa, T. hegewishii, and Glaucoma chattoni were cloned, sequenced, and compared with the previously cloned RNA gene from T. thermophila and with each other. To define secondary structures of these RNAs, homologous complementary sequences were identified by the occurrence of covariation among putative base pairs. Although their primary sequences have diverged rapidly overall, a strikingly conserved secondary structure was identified for all these telomerase RNAs. Short regions of nucleotide conservation include a block of 22 totally conserved nucleotides that contains the telomeric templating region. Introduction Telomeres, the specialized DNA-protein structures found at the ends of eukaryotic chromosomes, have been shown to be necessary for chromosome stability and the complete replication of chromosomal termini (reviewed in Blackburn and Szostak, 1984; Zakian, 1989). Telomeric DNA consists of a variable number of short, tandemly repeated G+C-rich sequences, one strand of which is G-rich. This strand is oriented 5' to 3' toward the chromosome terminus. The ribonucleoprotein enzyme telomerase synthesizes the G-rich strand of telomeric DNA. Telomerase activity was first identified in the holotrichous ciliate Tetrahymena thermophila (Greider and Blackburn, 1985) and has subse-' quently been found in the hypotrichous ciliates Euplotes crassus (Shippen-Lentz and Blackburn, 1989) and Oxytrichanova,(Zahler and Prescott, t988) as welt as" in human tissue culture cells (Morin, 1989). Telomerase adds species-specific telomeric repeats to the 3' end of G-rich telomeric DNA oligonucleotides in vitro. The RNA components of telomerase from Tetrahymena and Euplotes have been identified and have been found to include sequences corresponding to the complement of their species-specific telomeric repeat (Greider and Blackburn, 1989; ShippenLentz and Blackburn, 1990). Site-directed mutagenesis of this sequence in the Tetrahymena telomerase RNA gene

(CAACCCCAA, complementary to the TTGGGG telomeric repeat) followed by overexpression of the mutated gene in transformed Tetrahymena cells results in the synthesis of telomeres in vivo whose sequence corresponds to that of the mutated template (Yu et al., 1990). In vitro experiments with Euplotes telomerase have established that a corresponding portion of the telomerase RNA (CAAAACCCCAAA) serves as the template for the synthesis of Euplotes-specific telomeric repeats (TTTTGGGG) (Shippen-Lentz and Blackburn, 1990). Thus telomerase represents an unusual reverse transcriptase: one that contains its own internal RNA template to direct DNA synthesis. It is not known whether telomerase RNA has a function(s) other than serving as a template. The catalytic properties of RNA have been established for the group I and group II self-splicing introns (Zaug and Cech, 1986; Michel et al., 1989), the tRNA-processing activity of the RNA component of RNAase P (Guerrier-Takada and Altman, 1984), and self-cleaving RNAs. By analogy to the self-splicing intron ribozyme of Tetrahymena(Zaug and Cech, 1986; reviewed in Cech, 1990), whose in vitro polymerization activity uses an internal RNA template, telomerase RNA could also contribute to the catalysis of DNA synthesis. Identification of functional domains of telomerase RNA, in addition to that of the template, requires knowledge of its structure. Phylogenetic sequence comparisons between homologous RNAs have proven useful in elucidating higher order RNA structure (Fox and Woese, 1975; Noller and Woese, 1981; James et al., 1988; Larsen and Zwieb, 1991). In this approach, complementary sequences identify helical regions in an RNA. Putative base pairings are revealed by seeking an equivalent pairing in homologous RNAs in which the sequences vary. The maintainance of base-pairing potential for putative helical regions in RNA homologs supports the existence of such a structural element, while the absence of the covariation of putative base pairs that maintain complementarity can argue against the existence of the putative helical structure in vivo (reviewed in Pace et al., 1989). With the exception of what has been functionallydefined as the telomeric templating region (Greider and Blackburn, 1989; Shippen-Lentz and Blackburn, 1990; Yu et al., 1990), the sequences of telomerase RNAs from T. thermophila (159 nucleotides) and E. crassus (approximately 190 nucleotides) have diverged to the extent that no overall sequence alignment or structural elements can he~identified with confidence. It was therefore-necessary to compare telomerase RNAsfrorn more closely related species. We determined the sequences of telomerase RNAs from six additional ciliates (five species of Tetrahymena and Glaucoma chattoni) that have the same telomeric TTGGGG repeat as T. thermophila (Katzen et al., 1981; Challoner and Blackburn, 1986). Here we present a strikingly conserved secondary structure for these telomerase RNAs that is revealed and supported by the covariation of paried residues.

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Figure 1. GenomicSouthern BlottingAnalysisof Telomerase RNA Genes Five microgramsof total DNA from six Tetrahymenaspp. and 10 p.g from G. chattoniweredigestedwith Hindlll,electrophoresedon 0.8% agarose, transferredonto a Nytran membrane, and hybridizedwith the nick-translated520 bp Dral fragment from pCG1 (Greider and Blackburn,1989),whichcontainsthe T. thermophilatelomeraseRNA gene.

Results Determination of Telomerase RNA Sequences We cloned and sequenced the telomerase RNA genes from six species in the tetrahymenine group of ciliates. Southern blot hybridizations probed with the cloned single-copy T. thermophila telomerase RNA gene (G reider and Blackburn, 1989) identified a single Hindlll restriction fragment in the genomic DNA of G. chattoni and the five Tetrahymena spp. examined (Figure 1). Similar results were obtained using several restriction enzymes, indicating that each species has a single macronuclear genomic copy of the telomerase RNA gene. Genomic DNA from the more distantly related ciliates E. crassus and Paramecium tetraurelia did not reveal any significant cross-hybridization with the T. thermophila telomerase RNA gene when probed under low stringency conditions (Shippen-Lentz and Blackburn, 1990; D. P. R. and E. H. B., unpublished data), The telomerase RNA genes from G. chattoni and three

species of Tetrahymena (T. malaccensis, T. pyriformis, and T. hegewishii) were cloned from size-selected libraries and sequenced. A comparison of telomerase RNA sequences for T. thermophila and these three additional Tetrahymena spp. revealed a conserved sequence at the 3' end of the putative telomerase RNAs. Also conserved between the Tetrahymena spp. and G. chattoni genes is an 8 nucleotide sequence approximately 55 nucleotides upstream of the 5' termini of the telomerase RN&s. Oligonucleotides corresponding to these conserved segments were used to amplify the corresponding genes from the genomic DNA of two additional Tetrahymena species, T. hyperangularis and T. pigmentosa, by the polymerase chain reaction (PCR) as described in Experimental Procedures. The T. hyperangularis and T. pigmentosa PCR products were also cloned and sequenced (see below). The sequence data generated were then used in an inverse PCR strategy (described in Experimental Procedures) to determine their complete coding sequences. Primary Sequence Comparisons The 5' termini of the six Tetrahymena spp. telomerase RNAs were mapped by primer extension of an oligonucleotide complementary to the highly conserved template region. Similarly, the 5' terminus of the G. chattoni telomerase RNA was determined by extension of a DNA primer complementary to the 3' end of that RNA (Figure 2). An alignment of homologous nucleotides in the seven telomerase RNA sequences is shown in Figure 3. In all seven species there is a block of complete identity between nucleotide positions 39 and 60, inclusive. This 22 nucleotide sequence includes a functional domain of telomerase RNA: the telomeric templating region (nucleotide positions 46 to 54; see Greider and Blackburn, 1989; Yu et al., 1990). A high percentage of nucleotides at the 3' end of the molecule is also highly conserved (28 of 44 nucleotides between nucleotide positions 127 and 170). The alignment presented in Figure 3 was largely dependent on these two highly conserved sequence elements, as well as secondary structural elements, described below,. Table 1 shows the similarity values and the absolute numbers of nucleotide differences in pairwise comparisons of the telomerase RNAs. The telomerase RNA sequence from G. chattoni is approximately equidistant from the RNAs from all the Tetrahymena spp. (similarities between 64.7% and 66.20). Consistent with the phylogeny derived from rRNA sequences (Sogin et al., 1986; Preparata et al., 1989), the Tetrahymena telomerase RNA sequences can be grouped into two sets: one consists of T. thermophila, T. malaccensis, and T. pyriformis, and the other of T. pigmentosa, T. hyperangularis, and T. hegewishii. The sequence upstream of the telomerase RNA coding region contains two absolutely conserved sequences (nucleotide positions -61 to -54 and -28 to -28 relative to the 5' terminus of the T. thermophila RNA, defined as the +1 position). The upstream regions are illustrated in Figure 4 and discussed below.

TelomeraseRNA SecondaryStructure 345

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Figure2. Mappingthe 5' Ends of TelomeraseRNAs (A) Primerextensionanalysisfrom the Tetrahymenaspp. indicated.The oligonucleotideprimerusedis complementaryto nucleotidepositions+39 to +59, inclusive.The sequencingladderis that of the clonedT. thermoph~atelomeraseRNA gene, mappingthe 5' termini of Tetrahymenaspp. telomeraseRNAs relativeto that of the T. thermophilaRNA (Greiderand Blackburn, 1989). (B) Primerextensionof G. chattoniRNA, using an oligonucleotidecomplementaryto nucleotidepositions+151 to +168, inclusive.The template for the sequencingreactionswas the clonedG. chattoni telomeraseRNA gene. I n f e r e n c e of S e c o n d a r y S t r u c t u r a l E l e m e n t s on S e q u e n c e Alignment

Based

Complementary regions common to all seven telomerase RNA sequences were identified by pairwise comparisons of sequences as described in Experimental Procedures. Putative base pairings were established by the detection of more than one, and usually several, pairs of compensatory base changes at a position. The resulting proposed secondary structures for telomerase RNAs from G. chattoni and representatives of the T. thermophila/T, pyriformis and T. hegewishii/'l', pigmentosa groups are shown in Figure 5. This analysis reveals a strikingly similar secondary structure for the telomerase RNA from Tetrahymena spp. and G. chattoni based on four helices whose lengths and' relative positions are highly conserved. These four helices (designated I, II, III, and IV, proceeding 5'to 3') are shown in Figure 5 and are compared among all seven telomerase RNAs in Figure 6. One of the helical structures (I) is in the form of a long-range base pairing, while the other three are hairpin loops. There is also a stretch of 37 to 38 nucleotides for which no higher order structure could be deduced, based on covariation of potential base pairs either within this stretch or within other regions of the molecule. This long region of unknown structure includes the telomeric template embedded in a stretch of totally conserved nucleotides.

Telomerase

R N A Helical S t r u c t u r e s

The lengths and relative positions of the four telomerase RNA helices are remarkably constant for all seven species. Helix I, formed by the long-range base pairing, is totally conserved between all Tetrahymena species (Figure 5) and was identified based on two compensatory base changes in the homologous structure from the G. chattoni sequence. This helical structure effectively separates the RNA into two unequal halves. The 5' half contains the 37 to 38 nucleotide stretch of unknown structure, including the totally conserved telomeric template region, and two hairpin loops (11and III). The 3' half consists of a single long stem-loop structure (IV, see Figures 5 and 6). In Tetrahymena spp., the 5 bp helix I is made up entirely of G-C pairs (5"CCCGC-3' paired to 5'-GCGGG-3'). Using the method described by Turner et al. (1988), its calculated AG value is - 7 . 8 kcal/mol. In Glaucoma, the homologous structure has two compensatory base changes (5'-CCUCC-3' paired to 5'-GGAGG-3'). The lowest free-energy calculation for this 5 bp helix is -6.4 kcal/mol, suggesting that it is slightly less stable than the Tetrahymena helix. However, there is the potential for two additional base pairs in the Glaucoma sequence that decrease the calculated AG to -9.9 kcal/ mol (5'-CCUCCUG-3' paired to 5'-CAGGAGG-3'). Although these two additional base pairs in helix I are not supported by covariation in any of the other telomerase RNAs, they have been incorporated into the model shown

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for Glaucoma because of the resulting increase in stability for this long-range base pairing. In addition, the nucleotide distance between helices I and III still falls within the range seen for the other species (between 3 and 5 nucleotides) with the inclusion of these base pairs in the s e c o n d a r y structure model for G. chattoni telomerase RNA. In helices III and IV, maintainance of continuous base pairing required that bulged residues be included in the model. Of particular note in helix IV is a 2 nucleotide GA bulge flanked by two G-C base pairs that are conserved in all seven species (Figure 6). Helix II is flanked by an

absolutely conserved direct repeat (CAUU), and the helix loops for s t e m - l o o p s III and IV contain totally conserved residues (Figure 5). Unusual base pairings (G-U, U-U, A-A, and G-A) occur in all three helices. Noncanonical base pairs are included in the secondary structure only when there have been at least two independent c o m p e n s a t o r y base ~hanges that establish canonical base pairs at those helical positions (Figure 6). An unusual aspect of helix IV is its primary sequence conservation relative to that of helices II and II1. Shortrange base pairings that create hairpin loops tend to be

Table 1. Similarity and Nucleotide Distance Data between Telomerase RNA Gene Sequences T. the T. thermophila : T. malaccensis T. pyriformis T. pigmentosa T. hyperangularis T. hegewishii Glaucoma chattoni

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67

The upper right half of the table gives similarity values H for all pairwise comparisons of the telomerase RNA sequences in Figure 2. H is defined as in Sogin et al. (1986), where H = ml(rn + u + g12), m is the number of sequence positions with matching nucleotides in the two sequences, u is the number of sequence positions with nonmatching nucleotides, and g is the number of sequence positions that have a gap in one sequence opposite a nucteotide in the other sequence. The absolute number of base changes and gapped positions is shown in the lower half of the table.

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We have cloned and sequenced the telomerase RNA genes from six tetrahymenine species. Inspection of sequences immediately upstream of the mapped 5' ends of the telomerase RNAs revealed two totally conserved elements. At nucleotide position -61 is an 8 nucleotide sequence (ACCCATAA), followed by a 4 nucleotide sequence (TTTA) at position -28, embedded in an otherwise highly diverged region. The same motifs are also present upstream of the E. crassus telomerase RNA coding sequence at nucleotide positions - 6 2 and -28, respectively (Shippen-Lentz and Blackburn, 1990). The distance of these two conserved elements from the mapped 5' termini of all these telomerase RNAs is quite precise, varying by no more than 1 nucleotide. A sequence element similar to the octamer noted above has been detected at approximately the same position upstream of the T. thermophila U snRNA coding sequences (Tani and Ohshima, 1991; H. Qrum and H. Nielsen, personal communication). Nuclear run-off experiments by Yu et al. (1990) demonstrated that the sensitivity of T. thermophila telomerase RNA transcription to ~-amanatin was the same as that of 5S rRNA transcription, suggesting that telomerase RNA is an RNA:polymerase 411transcript. The sequences of, all eight telomerase RNA genes sequenced thus far have a stretch of between five and eight T residues at the end of the transcription unit. These properties are characteristic of RNA polymerase III transcription termination sites (Bogenhagen and Brown, 1981; Allison and Hall, 1985). A region of similarity to the box A consensus, an internal promoter element characteristic of RNA polymerase III genes (reviewed in Geiduschek and Tocchini-Valentini, 1988), was noted previously for the T. thermophila telomerase RNA (Greider and Blackburn, 1989). However, no

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obvious box A was identified in the E. crassus telomerase RNA (Shippen-Lentz and Blackburn, 1990), and none was detected in the additional telomerase RNA sequences presented here. Give the absence of any identifiable internal promoter elements for telomerase RNAs, we propose that the upstream sequences we have identified are promoter elements like those of vertebrate and Arabidopsis thaliana U6 snRNA genes (Mattaj et al., 1988; Lobo and Hernandez, 1989; Carbon et al., 1987; Waibel and Filipowicz, 1990). Telomerase RNA Sequences The evolutionary relatedness of many ciliated protozoans has been quantitated on the basis of rRNA sequences (Sogin et al., 1986; Preparata et al., 1989: Greenwood et al., 1991), and these data were a major consideration in our selection of organisms for a comparative structure analysis of telomerase RNAs. Sequence similarities of 6 0 % - 8 0 % are optimal for the identification of homologous secondary structures as revealed by the covariation of base pairs (Pace et al., 1989). A pairwise comparison of tetrahymenine telomerase RNA primary sequences showed similarities that range between 64.7% and 99.4%, representing a maximum of 69 changes out of 170 nucleotide positions (Table 1). In contrast, a pairwise comparison of 5.8S rRNA sequences from the same species reveals no more than 2 changes out of 154 nucleotides (Preparata et al., 1989), and the overall rate of divergence for telomerase RNA is at least one order of magnitude greater than that for 17S rRNA (compare Table 1 and Sogin et al., 1986).

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The 22 nucleotide tract that includes the telomeric ternplating domain is absolutely conserved in the tetrahymenine telomerase RNA sequences. The first 5 nucleotides of this tract match a 5 nucleotide sequence in an exactly corresponding position 5' of the template in the E. crassus telomerase RNA (Shippen-Lentz and Blackburn, 1990). Telomerase RNA Secondary Structure Even though primary sequence similarities among telomeras~ RNAs are as low as 65.2% between species, the overall secondary structure of the molecule is remarkably conserved (Figure 5). The lengths of the helices and helical loops are very constant, differing by no more than 2 nucleotides. The distances between helices are also highly conserved, most notably the 37 to 38 nucleotide region of unknown structure between helices II and III that includes the template domain. The unpaired nature of this region is supported by earlier experiments in which oligonucleotides complementary to this region in the telomerase ribonucleoprotein complex were tested for their ability to direct cleavage by RNAase H. The results of these experiments established that the template and the region immediately 3' of the templating domain are accessible to hybridization (Greider and Blackburn, 1989). The absence of secondary structure implies an inherent flexibility in this portion of the telomerase RNA that may be critical to the current model for the mechanism of telomeric DNA synthesis by telomerase (Shippen-Lentz and Blackburn, 1990; reviewed in Blackburn, 1991).

TelomeraseRNA SecondaryStructure 349

The constancy of the secondary structure model may reflect the narrow range of sizes for the tetrahymenine telomerase RNAs, all of which are between 155 and 161 nucleotides long. It is possible that all or part of this model represents a common core of primary and secondary structure for telomerase RNA. While the longer (192 nucleotide) telomerase RNA from E. crassus (ShippenLentz and Blackburn, 1990) can be drawn to include such a core, determination of its structure will require further phylogenetic sequence comparisons. The helical regions of telomerase RNA were identified by the covariation of homologous base pairs. Although this criterion was consistently applied in our analysis, each of the four helices has a distinctive character. Helix I involves long-range base pairing and is by far the most highly conserved of the helices. Identification of this interaction is critical, since it establishes the overall superstructure of the RNA. Helices II, III, and IV are all stem-loop structures, each with distinct attributes other than their obviously differing sizes and positions. Stem-loop II is well defined by covariation and by its position relative to a conserved 4 base direct repeat (CAUU) at the base of the stem. Though the lengths of the stem and loop are absolutely conserved, the sequences of the 5 nucleotide loop and the distal region of the helix vary at every position, indicating the importance of the secondary structure of helix II over its primary sequence. Conservation of stem-loop structures, and not necessarily of primary sequence, is often indicative of protein-binding domains, as in the binding site for ribosomal protein L11 in Escherichia coli 23S rRNA (reviewed in Draper, 1990). The first 2 nucleotide pairs at the base of stem-loop II are invariant, and so their inclusion in the helix is tentative. In contrast to helix II, there are totally conserved nucleotides in the loops of helices III and IV (Figure 6). An interesting aspect of stem-loop IV is the high degree of primary sequence conservation of base-paired nucleotides relative to that seen for stem-loops II and III (see Figure 6). This suggests that portions of the primary sequence of helix IV are as important as its secondary structure. Alternatively, lack of compensatory base pair changes could imply that the secondary structure is critical and that single base pair changes that disrupt this structure were not tolerated even transiently in evolution. The primary sequence conservation of helix IV relative to the other telomerase, RNA stem-loops is reminiscent of the region of U6 snRNA that forms a paired structure with the 5' end of U4 snRNA (reviewed in Guthrie and Patterson, 1988). Phylogenetic sequence comparisons reveal that while intramolecular U4 and U6 stem-loops are supported by covariation of base pairs at nearly every position in the helices, the nucleotides contributing to the two U4/U6 intermolecular stems are highly conserved. Mutagenesis of certain of these nucleotides shows that conservation of the U6, but not the U4, primary sequence is required for function (Madhani et al., 1990). It has been suggested that this sequence conservation may be indicative of a catalytic function of U6 snRNA in the splicing reaction (Guthrie, 1991). The high degree of primary sequence conservation of telo-

merase RNA helix IV may be a clue to its possible functional role (discussed below).

Telomere Elongation and Telomerase RNA Conformation Telomerase has been shown to be a processive enzyme in vitro (Greider and Blackburn, 1989; Greider, 1991). Elongation of a single-stranded DNA primer by telomerase in a processive manner implies a translocation event in which the 3' end of the newly synthesized telomeric repeat dissociates from the templating region to be repositioned for subsequent additions. Also implicit in such a mechanism are at least two different extreme conformations: the conformation just before translocation, and the conformation immediately afterward. Before translocation, the newly added telomeric repeat and the templating region of the telomerase RNA are inferred to constitute a 6 to 9 bp RNA-DNA heteroduplex. This transitory heteroduplex seems likely to affect the overall conformation of telomerase RNA. For example, it could participate in a coaxial stack with stem-loop II. A critical event in telomerase translocation is inferred to be the destabilization of the RNA-DNA helix and the subsequent dissociation of the heteroduplex. In vitro, the newly synthesized telomeric repeat is then free to reassociate with the 3' end of the ternplating domain to prime a continuation of telomeric DNA synthesis. It might be expected that the unwinding of the RNA-DNA heteroduplex prior to translocation is an energyrequiring step. Conformational changes in the RNA and/ or the telomerase proteins produced during DNA polymerization could build up conformational strain (using the triphosphate bond energy of the polymerizing nucleotides) and contribute to the destabilization of the RNA-DNA heteroduplex prior to the translocation step. Alternatively, although ATP and/or GTP is not required for telomerase activity in vitro (Greider and Blackburn, 1985, 1987), a DNA-RNA helicase activity that uses dGTP or d'l-I'P hydrolysis could be associated with or intrinsic to telomerase. The U4/U6 snRNA interaction appears to be dynamic and may undergo a cyclic dissociation during the course of spliceosome assembly and/or activity (Cheng and Abelson, 1987; Lamond et al., 1988). Conformational changes in 7SL RNA have been implicated in the progression of the signal recognition particle cycle during protein translocation (Andreazzoli and Gerbi, 1991). By analogy, it is possible that the totally conserved nucleotides of telomerase RNA helix IV could be involved in alternative base pairing that might help mediate a conformational change during a translocation event. One feature of helix IV is the presence of a totally conserved 3 nucleotide direct repeat (AUGGAUG). These same 7 nucleotides in helix IV are involved in base pairing that defines a totally conserved GA bulge loop (Figure 6). It has been shown that bulged loops tend to increase the number of conformations available to a helix and have an effect on helix stabilitythat may be propagated for several base pairs 0Nhite and Draper, 1989; Longfellow et al., 1990). A second totally conserved 3 nucleotide direct repeat that flanks the base of stemloop II (CAUU-CAUU) is complementary to the helix IV

Cell 350

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Figure 7. Alternative Base-Pairing Configurations for Telomerase RNA This schematic of the secondary structure model of T. thermophila telomerase RNA includes a template RNA-telomeric DNA heteroduplex (5'-. . . . TTGG,GGTIG-3~, and is drawn with helix II in an opposite orientation from that shown in Figure 5 for visual ease. The directionality (5' to 3') of two totally conserved and complementary direct repeats flanking helix II and included in helix IV is indicated by the black and white arrows, respectively. Various possible combinations of alternative base-pairing configurations involving conserved nucleotides (boldface type) are indicated by gray arrows. See text for discussion.

direct repeat (Figure 5). Base pairing, possibly involving base triples, by the helix IV repeats can be envisaged between either one or both repeats flanking helix I1. We note that nucleotides forming base triples in tRNAs are highly conserved (Tinoco et al., 1990; Sampson et al., 1990). A summary of potential base-pairing combinations involving the above-mentioned 3 nucleotide repeats is shown schematically in Figure 7. Any one of these five potential alternative base-pairing configurations would result in a pseudoknot structure (reviewed in Chastain and Tinoco, 1991) and could contribute to a coaxial stack that potentially could include helix II and an RNA t e m p l a t e telomeric DNA heteroduplex prior to translocation. The orientation and magnitude of this coaxial stack would differ considerably depending on which of the two repeat sequences flanking s t e m - l o o p II was base paired. The alternative base pairings outlined in Figure 7 involve absolutely conserved nucleotides exclusively. We stress that in the absence of covariation, there is no evidence for the existence of the alternative structures, only for the possibility of their occurrence. The derivation of secondary structures by phylogenetic comparative analysis of other RNAs, such as rRNAs (Noller and Woese, 1981), group I (Davies et al., 1982) and group II (Michel and Dujon, 1983)introns, RNAase P RNA (James et al., 1988), and spliceosomal snRNAs (reviewed in Guthrie and Patterson, 1988), has helped to identify invariant structures that have subsequently been implicated in catalytic sites. The total conservation of nucleotides is often an indication of structural and functional importance, as has been demonstrated dramatically for rRNAs (Noller et al., 1990). In light of the many examples of catalytic RNAs that have been discovered in the last decade (reviewed in Cech and Bass, 1986; Cech, 1990), there exists the distinct possibility that telomerase RNA is involved in not only the templating but also the catalysis of telomeric DNA synthesis. We have shown that the overall

secondary structure of the tetrahymenine telomerase RNA is highly conserved and that the telomeric templating domain is included in a region of 22 absolutely conserved nucleotides. With a proposed structure for telomerase RNA, we can begin to test for an additional functional role(s) of the RNA moiety by the same experimental approaches used to define the templating domain (Yu et al., 1990). Experimental Procedures General Methods

Restriction enzymes, T4 DNA ligase, and E. coil DNA polymerase I were obtained from New England BioLabs and used following the procedures of Sambrook et al. (1989). Avian myeloblastosis virus reverse transcriptase was purchased from Boehringer Mannheim. PCR reactions were carried out on a DNA Thermal Cycler with Taq DNA polymerase and PCR reagents from Perkin-Elmer Cetus. Oligonucleotides were radiolabeled at the 5' end with T4 polynucleotide kinase and [7-32P]ATP(ICN, specific activity 7000 Ci/mmol) as described in Samb.rooket al. (f 989). Cloning into pUC118 and pUC119 and preparation of double-stranded and single-stranded DNA from the appropriate E. coil host strains were as described (Vieira and Messing, 1987). Tetrahymena spp. were cultured as described (Yu and Blackburn, 1990). G. chattoni was maintained in ATCC medium 802, bacterized with Klebsiella pneumoniae (Daggett and Nerad, 1989). Hybridization Probe for Telomerase RNA Genes

A 520 bp Dral fragment from pCG1 containing the T. thermophila telomerase RNA gene (Greider and Blackburn, 1989) was gel purified from a 0.80/0 low melting agarose gel. The DNA was recovered from the gel slice by multiple freeze-thawing cycles, phenol extraction, and ethanol precipitation. Routinely, 0.3 to 0.6 pg of this fragment was nick translated to a specific activity of 2 x 107 to 4 x 107 cpm/pg with 60 pCi of [Q-32P]dNTPs(Amersham, specific activity 400 Ci/mmol) as described by Sambrook et al. (1989). DNA Isolation and Southern Blots

Total DNA was isolated from T. thermophila (strain SB 2120), T. malaccensis (strain MP 75), T. pyrifol'mis (strain ST), T. pigmentosa (strain UM 1060),T. hyperangularis (strain EN 10), T. hegewishii (strain KP7), and G, chattoni (strain GT 1) as described by Yu and Blackburn (1990). DNA was digested with restriction endonucleases (New England BioLabs); the DNA fragments were electrophoresed in a 0.8% agarose

Telomerase RNA Secondary Structure 351

gel and transferred to a Nytran filter (Schleicher and Schuell) in a vacuum blotting apparatus (LKB). The filter was incubated in hybridization buffer containing 30°/o (v/v) formamide, 10o/o dextran sulfate (500,000 MW), 5% SDS, 4 x SSC (0.6 M NaCI, 60 mM sodium citrate), 1 x Denhardt's solution, 25 mM sodium phosphate (pH 6.5), 10 mM EDTA, and 0.25 mg/ml high molecular weight RNA with 1 x 107 to 2 x 107 cpm of T. thermophila-specific probe per hybridization overnight at 30°C. Blots were washed at 30°C for 15 rain in 2 x SSC, 0.1% SDS twice, followed by a final wash with 1 x SSC, 0.1o/o SDS under the same conditions. Cloning and Sequencing Telomerase RNA Genes Preparative Hindlll digests of 30 to 40 pg of total DNA were electrophorased in a 0.8% low melting agarose gel, and, based upon previous Southern blot information, the regions containing the telomerase RNA genes were excised from the gel. DNA was recovered from the gel slice as described above for nick-translated probe production. The size-enriched DNA' was then cloned into the Hindlll site of pUC118. Colony hybridizations were performed as described by Sambrook etal. (1989), with the same probe and conditions used for Southern hybridizations. Fragments from each of the genes were subcloned into the appropriate polycloning sites of pUC118 and pUC119. The complete sequences of both strands of the subclonas were determined using the dideoxynucleotide-terminated method (Sanger et al., 1977) with Sequenase (USB) and [cz-~S]dATP (Amersham, 1000 Ci/mmol). PCR Amplification of Telomerase RNA Genes The telomerase RNA genes from T. pigmentosa and T. hyperangularis were amplified by PCR using the two oligonucleotide primers 5'-CGCCAGCTGCAG(AT)(AG)A(AG)ACCCATAA-3' and 5'-GCGCATCGATAAAAATATAGACATCCATTG-3' (Saiki et aL, 1988); 40 cycles were performed (denaturing 94°C for 1 rain, annealing 46°C for 2 min, extension 72°C for 3 min), finishing with 72°C for 10 min. Specific amplification of the telomerase RNA genes with these primers yields products approximately 245 bp long. An aliquot of the PCR products was electrophoresed in a 2% agarose gel and transferred to a Nytran filter as described above for Southern blot analysis. The blot was probedwith 1 x 107cpmofaradiolabeledoligonucleotidecomplemen tary to the telomerase RNA templating domain (5'-GCGAATTCTAGATI I I I GGGGTTG-3', specific activity 4 mCi/nmol), which hybridized to a single fragment at 250 bp for both T. pigmentosa and T. hyperangularis PCR products (data not shown). The oligonucleotide primers were designed with Pstl and Clal restriction sites at their 5' ends, which facilitated cloning of the PCR products into the Pstl and Accl polycloning sites of pUC118. DNA from six individual clones per ligation were sequenced as described above; all products gave identical sequence for each cloning (Figure 2). To determine the complete sequences of the telomerase RNA genes from T. pigmentosa and T. hyperangulalis, an inverse PCR strategy was used. Using the sequence information determined above, for each telomerase RNA sequence a pair of unique synthetic DNA oligonucleotides was used to prime synthesis from genomic DNA that had been digested with Dral and then circularized in vitro. In this protocol, one primer (minus strand) of the pair primed synthesis toward the 5' end of the coding region and into the upstream sequence, and the other primer (plus strand) primed toward the 3' end of the gene and into its, downstream region. For each reaction, 0.1-0.5 p,g of genomic DNA was restricted with Dral, which had been shown by Southern blotting to produce a 0.4 kb genomic fragment containing the entire RNA coding sequence. The DNA was diluted to ,'~1 pg/ml to favor intramolecular ligation, and ligated with 0.1 "U/pl of T4 ligase (BRL) at 16°C for 20 hr. One-tenth of the ligated DNA was subjected to PCR in a 100 pl reaction containing 0.2 mM dNTPs, 0.2 p.g of each primer, and 2.5 U of Taq polymerase (Perkin-Elmer). The primer oligonculeotide sequences used corresponded to +52 to +34 (minus strand) and +58 to +76 (plus strand) for the T. pigmentosa and T. hyperangularis genes. Beginning with 94°C for 2 min, 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s were performed, finishing with 72°C for 10 min. The 0.4 kb PCR products visible by ethidium bromide staining after agarose gel electrophoresis were identified as the expected products by digestion with restriction enzymes Bglll and BamHI, which gave the predicted restric-

tion fragments. The PCR products were subsequently cloned and sequenced as described above. RNA Isolation and Primer Extensions Logarithmically growing 50 ml cultures (approximately 5 x 104 cells per ml) were pelleted at room temperature and immediately lysed, and total RNA was extracted by the method of Strohman et alo (1977) with modifications. Pelleted cells were resuspended quickly in 4.5 ml of lysing medium (DEPC-treated 6 M guanidinium HCI, 0.1 M potassium acetate [pH 5.0]) at -20°C. The lysate was precipitated with 1/2 vol of 100% ethanol at -20°C for a minimum of 1 hr. The mixture was centrifuged for 20 rain at 5000 rpm (Sorvall HB-4 rotor) at -20°C, and the pellet drained dry and rasuspended in 2.5 ml of 6 M guanidinium HCI, 25 mM EDTA (pH 7.0) at room temperature. After gentle vortexing, 2 M potassium acetate (pH 5.0) was added to a final concentration of 0.1 M, and the solution was reprecipitated with 1/2 vol of 100% ethanol at - 2 0 ° C for 12 hr. The mixture was centrifuged for 20 min at 5000 rpm (Sorvall HB-4 rotor) at -20°C; the pellet was rasuspended in 0.3 ml of 20 mM EDTA (pH 7.0) and extracted with an equal volume of phenoI-CHCI3 (1:1). After a brief centrifugation for phase separation, the organic phase was rsextracted with an additional 0.1 ml of 20 mM EDTA (pH 7.0). The aqueous phases were pooled, extracted with an equal volume of CHCIs, and precipitated by the addition of 2 M sodium acetate (pH 4.5) to a final concentration of 0.2 M and 2 vol of 100% ethanol (at - 2 0 ° C for a minimum of I hr). The precipitate was pelleted, washed once with 70% ethanol, and resuspended in double-distilled water to a final concentration of approximately 4 mg/ml, as indicated by ~ (Sambrook et al., 1989). Primer extension reactions were essentially as previously described (McKnight and Kingsbury, 1982). Approximately 2 pmol of a radiolabeled oligonucleotide primer complementary to the templating region of Tetrahymena spp. telomerase RNA (5'-GCGAATTCTAGAI I II IGGGGI-IG-3', specific activity 4 mCi/nmol) was used in primer extensions of Tetrahymena spp. RNAs. Similarly, a primer complementary to the 3' end of G. chattoni telomerase RNA (5'-CAGCTGATCAACTTGGCATTCCATAAG-3', specific activity 4 mCi/nmol) was used in primer extensions of G. chattoni total RNA. RNA Secondary Structure The initial analysis was augmented by a computer program that predicts RNA folding based on lowest free-energy estimates of helix stability (Zuker, 1989). Given the accessibility of the telomeric templating region (CAACCCCAA) to binding by telomeric and complementary antisense DNA oligonucleotidas in vitro (Greider and Blackburn, 1989; M. Lee and E. H. B., unpublished data), we imposed the constraint that these 9 nucleotides were unpaired. The resulting thermodynamically predicted secondary structures were used in conjunction with visual inspection of the sequence alignment to identity potential complementary segments. In particular, conserved nucleotides flanking the putatively base-paired regions and those occurring in helical loops were taken into account in assigning structures. Thus, the assignment of stem-loops is dependent on context (the relative positions of highly conserved, and therefore homologous, flanking sequences and hairpin loops) as well as on the presence of compensatory base changes (Pace et al., 1989). Acknowledgments We thank M. Chastain for his contribution in RNA secondary structure predictions based on lowest free-energy computations, H. Nielsen for assistance in the identification of conserved upstream sequences, and N. Pace, C. Guthrie, I. Tinoco, and members of the Blackburn laboratory for critical discussions of the manuscript, This work was supported by United States Public Health Service Grant GM 26259 to E. H. B. and a University of California President's postdoctoral fellowship to D. P. R. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received May 29, 1991; revised July 30, 1991.

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References Allison, D. S., and Hall, B. D. (1965). Effects of alterations in the 3' flanking sequence on in vivo and in vitro expression of the yest SUP4-o tRNA TM gene. EMBO J. 4, 2657-2664. Andreazzoli, M., and Gerbi, S. A. (1991). Changes in 7SL RNA conformation during the signal recognition particle cycle. EMBO J. 10, 767777. Blackburn, E. H. (1991). Structure and function of telomeres. Nature 350, 569-573. Blackburn, E. H., and Szostak, J. W. (1964). The molecular structure of centromeres and telomeras. Annu. Rev. Biochem. 53, 163-194. Bogenhagen, D. F., and Brown, D. D. (1981). Nucleotide sequences in Xenopus 5S DNA required for transcription termination. Cell 24, 261-270. Brow, D. A., and Guthrie, C. (1988). Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature 334, 213-218. Brow, D. A., and Guthrie, C. (1990). Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes Dev. 4, 1345-1356. Carbon, P., Murgo, S., Ebel, J.-P., Krol, A., Tebb, G., and Mattaj, I. W. (1967). A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase I1. Cell 51, 71-79. Cech, T. R. (1990). Self-splicing of group I introns. Annu. Rev. Biochem. 59, 543-568. Cech, T. R., and Bass, B. L. (1986). Biological catalysis by RNA. Annu. Rev. Biochem. 55, 599-629. Challoner, P. B., and Blackburn, E. H. (1986). Conservation of sequences adjacent to the telomeric CJ~ repeats of ciliate macronuclear ribosomal RNA gene molecules. Nucl. Acids Res. 14, 6299-6311. Chastain, M., and Tinoco, I., Jr. (1991). Structural elements in RNA. Prog. Nucl. Acids Res. Mol. Biol. 41,131-177. Cheng, S.-C., and Abelson, J. (1987). Spliceosome assembly in yeast. Genes Dev. 4, 1014-1027. Daggett, P.-M., and Nerad, T., eds. (1989). Supplement to: American Type Culture Collection Catalogue of Protists--Algae/Protozoa (Rockville, Maryland: American Type Culture Collection). Davies, R. W., Waring, R. B., Ray, J. A., Brown, T. A., and Scazzocchio, C. (1982). Making ends meet: a model for RNA splicing in fungal mitochondria. Nature 300, 719-724. Draper, D. E. (1990). Structure and function of ribosomal proteinRNA complexes: thermodynamic studies. In The Ribosome: Structure, Function and Evolution, W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner, eds. (Washington, DC: American Society for Microbiology), pp. 160-167. Fox, G. E., and Woese, C. R. (1975). 5S RNA secondary structure. Nature 256, 505-507. Geiduschek, E. P., and Tocchini-Valentini, G. P. (1988). Transcription by RNA polymerase III. Annu. Rev. Biochem. 57, 873-914. Greenwood, S. J., Schlegel, M., Sogin, M. L., and Lynn, D. H. (1991). Phylogenetic relationships of Blepharisma americanum and Colpoda inflate within the phylum Ciliophora inferred from complete small subunit rRNA gene sequences. J. Protozool. 38, 1-6. Greider, C. W. (1991). Telomerase is processive. Mol. Cell. Biol. 11, 4572--4580. Greider, C. W., and Blackburn, E. H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405-413. Greider, C. W., and Blackburn, E. H. (1987). The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 51,887-898. Greider, C. W., and Blackburn, E. H. (1989). A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331-337. Guerrier-Takada, C., and Altman, S. (1984). Structure in solution of M1 RNA, the catalytic subunit of RNAse P from Escherichia coll. Biochemistry 23, 6327-6334.

Guthrie, C. (1991). Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science 253, 157-163. Guthrie, C., and Patterson, B. (1988). Spliceosomal snRNAs. Annu. Rev; Genet. 22, 387-419. James, B. D., Olsen, G. J., Liu, J., and Pace, N. R. (1988). The secondary structure of ribonuclease P RNA, the catalytic element of a ribonucleoprotein enzyme. Cell 52, 19-26. Katzen, A. L., Cann, G. M., and Blackburn, E. H. (1981). Sequencespecific fragmentation of macronuclear DNA in a holotrichous ciliate. Cell 24, 313-320. qlw Lamond, A. I., Konarska, M. M., Grabowski, P. J., and Sharp, P. A. (1988). A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Proc. Natl. Acad. Sci. USA 85, 411-415. Larsen, N., and Zwieb, C. (1991). SRP-RNA sequence alignment and secondary structure. Nucl. Acids Res. 19, 209-215. Lobo, S., and Hernandez, N. (1989). A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase tll promoter. Cell 58, 55-67. Longfellow, C. E., Kierzek, R., and Turner, D. H. (1990). Thermodynamic and spectrophotometric study of bulge loops in oligoribonucleotides. Biochemistry 29, 278-285. Madhani, H. D., Bordonne, R., and Guthrie, C. (1990). Multiple roles for U6 snRNA in the splicing pathway. Genes Dev. 4, 2264-2277. Mattaj, I., Dathan, N., Parry, H., Carbon, P., and Krol, A. (1988). Changing the RNA polymerase specificity of U snRNA gene promoters. Cell 55, 435-442. McKnight, S. L., and Kingsbury, R. (1982). Transcriptional control signals of a eukaryotic protein-coding gene. Science 217, 316-324. Michel, F., and Dujon, B. (1983). Conservation of secondary structures in two intron families including mitochondrial-, chloroplast- and nuclear-encoded members. EMBO J. 2, 33-38. Michel, F., Umesono, K., and Ozeki, H. (1989). Comparative and functional anatomy of group II catalytic introns-a review. Gene 82, 5-32. Morin, G. B. (1989). The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59, 521529. Noller, H. F. (1984). Structure of ribosomal RNA. Annu. Rev. Biochem. 53, 119-162. Noller, H.. F., and Woese, C. R. (1981). Secondary structure of 16S ribosomal RNA. Science 212, 403-411. Noller, H. F., Moazed, D., Stern, S., Powers, T., Allen, P. N., Robertson, J. M., Weiser, B., and Timan, K. (1990). Structure of rRNA and its functional interactions in translation. In The Ribosome: Structure, Function and Evolution, W. E. Hill, A. Dahlberg, R. A. Garrett, P. B. Moors,.D. Schlessinger, and J. R. Warners, eds. (Washington, DC: American Society for Microbiology), pp. 73-92. Pace, N. R., Smith, D. K., Olsen, G. J., and James, B. D. (1989). Phylogenetic comparative analysis and the secondary structure of ribonuclease P RNA--a review. Gene 82, 65-75. Preparata, R. M., Meyer, F. P., Simon, E. M., Vossbrinck, C. R., and Nanney, D. L. (1989). Ciliate evolution: the ribosomal phylogenies of the tetrahymenine ciliates. J. Mol. Evol. 28, 427-441. Roiha, H., Shuster, E. O., Brow, D. A., and Guthrie, C. (1989). Small nuclear RNAs from budding yeast: phylogenetic comparison reveal extensive size variation. Gene 82, 137-144. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, second edition (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Sampson, J. R., DiRenzo, A. B., Behlen, L. S., and Uhlenbeck, O. C. (1990). Role of the tertiary nucleotides in the interaction of yeast phenylalanine tRNA with its cognate synthetase. Biochemistry 29, 25232532. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing

Telomerase RNA Secondary Structure 353

with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 54635467. Shippen-Lentz, D., and Blackburn, E. H. (1989). Telomere terminal transferase activity in the hypotrichous ciliate Euplotes crassus. Mol. Cell. Biol. 9, 2761-2764. Shippen-Lentz, D., and Blackburn, E. H. (1990). Functional evidence for an RNA template in telomerase. Science 247, 546-552. Siliciano, P. G., Brow, D. A., Roiha, H., and Guthrie, C. (1987). An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-1ike snRNA. Cell 50, 585-592. Sogin, M. L., Ingold, A., Karlok, M., Nielsen, H., and Engberg, J. (1986). Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups. EMBO J. 5, 3625-3630. Strohman, R. C., Moss, P. S., Micou-Eastwood, J., Spector, D., and Przybyla, A. (1977). Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures. Cell 10, 265-273. Tani, T., and Ohshima, Y. (1991). The genes for U6 small nuclear RNA in Tetrahymena thermophila are repeated in tandem. Nucl. Acids Res. 19, 2495. Tinoco, I., Jr., Publisi, J. D., and Wyatt, J. R. (1990). RNA folding. In Nucleic Acids and Molecular Biology, volume 4, F. Eckstein and D. M. J. Lilley, eds. (New York: Springer-Verlag), pp. 205-226. Turner, D. H., Sugimoto, N., and Freier, S. M. (1988). RNA structure prediction. Annu. Rev. Biophys. Chem. 17, 167-192. Vieira, J., and Messing, J. (1987). Production of single-stranded plasmid DNA. Meth. Enzymol. 153, 3-11. Waibel, F., and FiUpowicz, W. (1990). U6 snRNA genes of Arabidopsis are transcribed by RNA polymerase III but contain the same two upstream promoter elements as RNA polymerase II-transcribed U-snRNA genes. Nucl. Acids Res. 18, 3451-3457. White, S. A., and Draper, D. E. (1989). Effects of single base bulges on intercalator binding to small RNA and DNA hairpins and a rRNA fragment. Biochemistry 28, 1892-1897. Yu, G.-L., and Blackburn, E. H. (1990). Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila. Mol. Cell. Biol. 10, 20702080. Yu, G.-L., Bradley, J. D., Attardi, L. D., and Blackburn, E. H. (1990). In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 344, 126-132. Zahler, A. M., and Prescott, D. M. (1988). Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for the replication of the ends of linear DNA molecules. Nucl. Acids Res. 16, 6953-6972. Zakian, V. A. (1989). Structure and function of telomeres. Annu. Rev. Genet. 23, 579-604. Zaug, A. J., and Cech, T. R. (1986). The intervening sequence of Tetrahymena is an enzyme. Science 231,470-475. Zuker, M. (1989). On finding all suboptimal foldings of an RNA molecule. Science 244, 48-52.