INTRODUCTION

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Telomerase is a ribonucleoprotein (RNP) that is responsible for maintaining the terminal repeats of telomeres in most organisms (15). It acts as an unusual reverse transcriptase (RT), using a small segment of an integral RNA component as template for the synthesis of the dGT-rich strand of telomeres (16).

Telomerase activity has been characterized from a wide range of organisms and genes encoding both the RNA and protein components of the enzyme complex identified (for reviews, see references 2 and 41). Telomerase RNAs found in ciliated protozoa, in addition to having a short templating region, share a common secondary structure. Telomerase RNAs from yeasts and mammals are considerably larger and exhibit no evident structural conservation. The catalytic RT protein subunit (TERT), initially purified from Euplotes aediculatus as p123, was subsequently found to be homologous to Est2p, a yeast protein required for telomere maintenance (25, 26, 28). Both proteins possess RT-like motifs, alterations in which led to inactivation of telomerase activity and reduced telomere length. Subsequently, homologs of TERT were identified in Schizosaccharomyces pombe, humans, mice, and Tetrahymena, Oxytricha, and Arabidopsis spp. (3, 6, 10, 13, 22, 33, 38, 42). An evident homolog can also be discerned in the incomplete Candida albicans database (see Materials and Methods). Additional mutational analysis of the RT motifs in these latter proteins supports a role for TERT in directly mediating catalysis (20, 50). Because coexpression of TERT and telomerase RNA in vitro in the rabbit reticulocyte lysate system suffices to reconstitute enzyme activity (1, 50), these two subunits probably constitute the core of the enzyme complex.

Several groups of telomerase-associated polypeptides have been identified using either biochemical or genetic tools. First, purification of the Tetrahymena telomerase complex led to the discovery of two associated polypeptides (p80 and p95). Cloning and characterization of p80 and p95 suggest that these proteins may interact with telomerase RNA and the DNA primer, respectively (8, 12). Mouse and human homologs of p80 have also been identified and been shown to associate with the respective telomerases (19, 39). Second, a significant fraction of the human telomerase complex is apparently associated with the molecular chaperones Hsp90 and p23, which are also necessary for reconstitution of telomerase in vitro activity in the rabbit reticulocyte lysate system (21). These molecular chaperones are hypothesized to play a role in telomerase biogenesis. Third, two Sm proteins that are necessary for snRNA maturation are also components of the yeast telomerase complex, suggesting a role for these factors in telomerase RNA processing and telomerase complex assembly (47). Finally, genetic analysis of yeast identified two Est proteins (Est1p and Est3p) that act in the same pathway as telomerase RNA and TERT. Subsequent characterizations indicate that Est1p and Est3p are also subunits of the telomerase complex and that Est1p may play a role in the recruitment of telomerase core to telomere ends in vivo (9, 25, 41, 49). The precise biochemical and physiologic functions of the telomerase-associated proteins remain to be elucidated. The stoichiometry of telomerase components in the native complex is unclear. However, recent studies suggest that the yeast complex may contain more than one copy of the RNA component (43).

Previous analysis of all of the cloned TERTs revealed several salient features of their structural organization: (i) all of the RT motifs are located in the C-terminal half of the protein; (ii) a telomerase-specific motif (T motif), located just N terminal to the RT motifs, can be discerned in all TERTs; and (iii) a motif positioned further toward the N terminus (CP motif) appears to be much more highly conserved among all the ciliate telomerases and may perform a function specific to ciliate telomere formation (3). Point mutations in conserved residues of the T motif significantly impaired telomerase function in the rabbit reticulocyte lysate system (50). Other N-terminal regions of the TERT polypeptide have not been subjected to detailed molecular analysis.

To determine if the uncharacterized, N-terminal regions of TERT are functionally important and if they played conserved roles in telomere maintenance, we initiated mutagenic analysis of the yeast TERT (Est2p). As a starting point for this analysis, we performed a hidden Markov model (HMM)-based alignment of all available TERTs, including the recently identified C. albicans homolog. Several earlier applications of the HMM approach have resulted in the detection of homologies between distantly related proteins (34, 35, 37). Interestingly, such an approach in the case of TERT led to the identification of four conserved motifs in the N-terminal, non-RT region. To validate the predictions arising from comparative sequence analysis, we introduced substitution mutations into the yeast TERT (EST2) gene and investigated their effects on growth, telomere maintenance, and telomerase activity. In this paper, we report that the most N-terminal motif, called GQ, is indeed functionally important. Characterization of mutant phenotypes suggests that the GQ motif may play at least two distinct functions. Furthermore, we show that a nonconserved region located N terminal to the GQ motif (which we call the N region) is also functionally important, possibly through interacting with some nucleic acid target in the context of the native RNP.

While this work was in progress, Friedman and Cech (11) reported the identification of essential domains located in the N-terminal region of yeast TERT using a unigenic evolution approach. In this approach, the gene of interest is heavily mutagenized, and functional variants are selected. Essential and dispensable regions of the protein are then identified by statistically analyzing the distribution of missense and silent mutations. The regions identified in both their and our studies are largely concordant, and we comment on the similarities and discrepancies in the Discussion section.

This work was supported by the American Cancer Society, the Concert for the Cure, and the AMDeC Foundation (N. F. Lue) and by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under contract no. DE-AC03-76SF00098 (I.S. Mian). Sequencing of C. albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund.

J.X. and Y.P. contributed equally to the work.

	MATERIALS AND METHODS

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Yeast strains and plasmids. The haploid Saccharomyces cerevisiae strain W303-a (MATa ade2-1 trp1-1 leu2-3,112 his3-11,15 ura3-1 can1-100) was used for the construction of the est2 strain. The disruption cassette was made by inserting ~600 to 800 bp of the est2 gene flanking sequence upstream and downstream of the kanamycin marker of pUG6. The resulting fragment replaced amino acids 50 to 690 of Est2p open reading frame with the kanamycin resistance marker. Following transformation and selection on G418-containing plates, deletion of the est2 gene in selected isolates was confirmed by PCR.

Sequence comparison. All sequences used in comparative analysis, with the exception of the C. albicans TERT homolog, were obtained from GenBank. Sequence data for C. albicans was obtained from the Stanford DNA Sequencing and Technology Center website at http://www-sequence.stanford.edu/group /candida.

Determination of telomere length. Chromosomal DNA was isolated using the Smash and Grab protocol, digested with XhoI or PstI restriction enzyme, and electrophoretically separated on a 1% agarose gel. Following capillary transfer to nylon membranes, telomere-containing fragments were detected by hybridization with a ³²P-labeled poly(dG-dT) probe.

Purification of and assay for yeast telomerase. Whole-cell extracts and active DEAE fractions were prepared as previously described (5, 29, 30). A typical telomerase reaction was carried out in 30 µl containing the following: 10 mM Tris-HCl (pH 8.0), 2 mM magnesium acetate, ~300 mM sodium acetate (contributed by the protein fraction), 1 mM spermidine, 1 mM dithiothreitol, 5% glycerol (contributed by the protein fraction), 50 µM dTTP, 20 µCi of [-³²P]dGTP (3,000 Ci/mmol), 5 µM primer oligodeoxynucleotides (TEL66 [30]), and 15 µl of column fractions. Primer extension products were processed and analyzed by gel electrophoresis as previously described (29, 30). Signals were quantified using a PhosphorImager system (Molecular Dynamics). For quantification of activity, the signals from all labeled and RNase-sensitive products are summed.

Western analysis of protein A-tagged Est2p. Depending on the expression level, the amount of protein A-tagged Est2p was analyzed either directly in crude extracts or following IgG-Sepharose precipitation using the ProtoBlot system (Promega). For analysis using crude extracts, ~300 to 500 µg of total protein was electrophoresed into a sodium dodecyl sulfate (SDS)-8% polyacrylamide gel and transferred to nitrocellulose membrane. Primary anti-protein A antibody (Sigma) and secondary antibody were used at 1:1,000,000 and 1:5,000 dilutions, respectively. The high primary antibody dilution was necessary to minimize background arising from cross-reacting polypeptides. For analysis using IgG-Sepharose-purified telomerase, ~5 mg of unfractionated extracts was treated with 40 µl of IgG-Sepharose at 4°C for 16 h. The beads were washed three times with TMG-10(600), and bound proteins were eluted with SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer. Following electrophoresis and blotting, Est2p was detected by using primary and secondary antibody at 1:20,000 and 1:5,000 dilutions, respectively.

Construction of Escherichia coli EST2 fusion protein expression plasmids and purification of fusion proteins from E. coli. Fragments of the EST2 gene were amplified by PCR and cloned between the BamHI and PstI sites of the pMAL-cri vector (New England Biolabs). In addition, the downstream primers all possessed sequences that encode an in-frame six-His tag, allowing the fusion proteins to be purified using both nickel-affinity and amylose-affinity chromatography. The proteins expressed from these plasmids are named MBP-Est2(1-160)p, etc., with the numbers in parentheses indicating the amino acid residues of Est2p included in the fusion protein.

Filter binding assay. Protein-nucleic acid binding assays were performed using a modification of a published procedure (4). Est2p fusion proteins (from 0.25 to 5 µg) were mixed with ³²P-labeled nucleic acid and 50 µg of bovine serum albumin in 10 mM Tris (pH 8.0) and 10% glycerol. The reaction mixtures (50-µl total volume) were incubated on ice for 30 min and pipetted over a prewetted nitrocellulose filter (BA85; Schleicher & Schuell) sandwiched in a slot blot apparatus. The reaction mixtures were then slowly filtered through the membrane using gentle suction. The filters were washed three times with 0.5 ml of buffer (10 mM Tris, 300 mM sodium acetate, 50 µg of bovine serum albumin), air dried, and exposed to a PhosphorImager screen (Molecular Dynamics). All assays were done in duplicate, and the signals were averaged for further analysis. The difference between duplicate samples was generally <10%.

	RESULTS

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HMM-based analysis revealed four conserved motifs located in the N-terminal, non-RT region of TERT. To determine if the N-terminal, non-RT regions of TERT are functionally important and if they play conserved roles in telomere maintenance, we initiated mutagenic analysis of Est2p (the yeast TERT). Several earlier applications of HMM-based sequence comparison resulted in detection of homologies between distantly related proteins (34, 35, 37). Therefore, as a starting point for our analysis, we performed an HMM-based alignment of all available TERTs, including recently identified Arabidopsis thaliana and C. albicans homologs. After obtaining an alignment of the entire amino-terminal region, we defined the motif-domain boundaries by visual inspection. The criteria used for assigning regions of the alignment as linkers included low compositional complexity, absence of clusters of conserved residues, and large numbers of gaps in the majority of sequences. Interestingly, this protocol identified four conserved motifs in the N-terminal, non-RT region of TERT (Fig. 1). We call these motifs in order from N to C terminus the GQ, CP, QFP, and T motifs, respectively. The locations of these motifs within Est2p are as follows: GQ, residues 45 to 163; CP, residues 245 to 265; QFP, residues 267 to 343; and T, residues 367 to 413. Each motif contains nearly invariant amino acid residues that are located at fixed distances from one another. The GQ and QFP motifs have not been previously recognized. In addition, the CP motif, hypothesized earlier to be ciliate specific, is shown in our analysis to possess invariant and nearly invariant residues. The identification of conserved motifs throughout the N-terminal region of TERT suggests that this region mediates a conserved function(s) in telomere synthesis. The overall comparison also revealed a particularly degenerate region that is variable in length, located between motifs GQ and CP, implying the presence of a flexible linker (Fig. 1B).

View larger version (135K): [in this window] [in a new window]   FIG. 1.   Identification of conserved motifs in the N-terminal, non-RT region of telomerase RT. (A) A schematic illustration of the locations of RT motifs in the telomerase RT polypeptide as determined by an earlier analysis is shown at the top (3). HMM-based analysis revealed four conserved motifs located in the N-terminal region of all TERTs that have been identified thus far (from Tetrahymena thermophila, Oxytricha trifallax, E. aediculatus, S. cerevisiae, S. pombe, C. albicans, Mus musculus, Homo sapiens, and A. thaliana). These motifs are named in order from N to C terminus the GQ, CP, QFP, and T motifs, respectively. For ease of discussion, we also designate the most N-terminal nonconserved region of TERT the N region. The segment between the GQ motif and the CP motif is particularly variable in length, consistent with the existence of a flexible linker in this region. (B) A detailed alignment of the four motifs is shown. The GQ motif is shaded yellow, and the CP, QFP, and T motifs are shaded gray. Invariant residues are italicized, and highly conserved residues are shown in red. Conserved hydrophobic residues are shown in boxes. Closed circles denote point mutations described in this study, and open triangles denote functional mutations at nonconserved positions as reported by Friedman and Cech (11). Functional amino acid substitutions at conserved positions are explicitly given at the bottom. The numbers in parentheses are the numbers of functional mutations in connecting regions. Regions I, II, III, and IV as defined by Friedman and Cech (11) are indicated at the top. Sequences are from T. thermophila, C. trifallax, E. aediculatus, S. cerevisiae, S. pombe, C. albicans, M. musculus, H. sapiens, and A. thaliana (top to bottom, respectively).

The GQ motif is required for telomere maintenance. To validate some of the predictions arising from comparative sequence analysis, we constructed mutants of the EST2 gene bearing alanine substitutions in conserved residues in the GQ motif. The mutated EST2 gene, located on a centromeric shuttle vector under the control of its natural promoter, was transformed into an est2::Kan^r strain that had been grown in the absence of Est2p for ~25 to 50 generations. The resulting strain was then monitored for defects in growth, telomere maintenance, and telomerase activity. To facilitate future biochemical analysis, the plasmid-borne EST2 gene was fused at its 3' end with three tandem Myc tags and a six-His tag. In some cases, an additional protein A tag (consisting of two copies of the IgG binding domain from protein A) was inserted in between the Myc and His tags to allow even more efficient affinity purification. These C-terminal modifications have no effect on telomere maintenance and telomerase activity (J. Xia, unpublished data).

View larger version (85K): [in this window] [in a new window]   FIG. 2.   Telomere length determination in strains that contain mutations in the conserved and nonconserved residues within the GQ motif. The est2 strain that had been grown for ~25 to 50 generations was transformed with plasmids bearing either wild-type (W.T.) or mutated EST2. The transformants were restreaked twice, and single colonies were picked for growth in liquid culture. Chromosomal DNAs were isolated from the cultures, digested with PstI, electrophoresed into a 1% agarose gel, transferred to a nylon membrane, and probed with 32P-labeled poly(dG-dT). The locations of the Y' telomeres and an X telomere are indicated by a vertical bar and an arrowhead, respectively. The mobilities of several molecular size standards are indicated on the left.

Two mutations impaired telomere maintenance without affecting telomerase activity. Because several studies point to the existence of mutations that uncouple in vitro telomerase activity from in vivo telomere maintenance (e.g., est1 and est3 mutants [27]), we were interested in determining if any of the GQ mutations had similar properties. Extracts were prepared from each of the mutant strains, and telomerase activity was partially purified over DEAE columns and tested. Earlier studies indicated that, under standard reaction conditions, this chromatographic fraction yields labeled products that are almost entirely due to TLC1 and Est2p (5; N. Lue, unpublished data). As shown in Fig. 3A, almost the entire primer extension signal in this preparation is sensitive to RNase A pretreatment. In addition, when the Est2p in the strain is tagged with two copies of the IgG binding domain from protein A, >80% of the activity in the DEAE fraction can be specifically trapped on IgG-Sepharose beads and depleted from supernatant, indicating that this fraction is largely free of other contaminating activities (Fig. 3A).

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Telomerase primer extension assays for wild-type and mutated RNPs. (A) The DEAE fraction was prepared from strains whose Est2p was either tagged or untagged with two copies of the IgG binding domain from protein A. The fractions were incubated either in the absence () or in the presence (+) of IgG-Sepharose beads. The supernatants were then recovered, incubated in the absence () or the presence (+) of RNase A, and assayed for telomerase activity using TEL66 (TAGGGTAGTAGTAGGG) as the primer oligonucleotide. The position of the primer +3 products is marked to the left of the panel. (B) DEAE fractions were prepared from strains bearing wild-type and mutated Est2p. Each mutant fraction was tested alongside the wild-type fraction using equal amounts of total protein. Each panel presents results from a single set of assays. The identities of the mutations are indicated at the top of each panel, and RNase-sensitive signals derived from telomerase are indicated by vertical bars to the left of each panel. An RNase-insensitive band (indicated by an asterisk) is occasionally observed in some assays.

The extreme N-terminal region of Est2p (N region) is also required for telomere maintenance in vivo and telomerase activity in vitro. Our comparative analysis suggests that the extreme N-terminal 50 amino acids (termed N region) of yeast TERT could not be reliably aligned with its homologs from other species. To test the importance of this N region, we constructed N-terminal truncation mutants of the EST2 gene and tested their ability to support telomere length maintenance and telomerase activity using the previously described system.

View larger version (47K): [in this window] [in a new window]   FIG. 4.   N-terminal deletions result in defective Est2p function. (A) Transformants bearing either wild-type or N-terminally deleted Est2p were restreaked twice and monitored for growth defects on minimal plates. The photographs show colonies from the second restreak after 2 days of growth. The identities of the clones are indicated at the left of each panel. (B) Transformants bearing either wild-type or N-terminally deleted Est2p were restreaked twice, and single colonies were picked for growth in liquid cultures. Chromosomal DNAs were isolated from the cultures, digested with XhoI electrophoresed into a 1% agarose gel, transferred to a nylon membrane, and probed with 32P-labeled poly(dG-dT). The locations of the Y' telomeres and X telomeres are indicated by a vertical bar and several arrowheads to the right of the panel, respectively. (C) DEAE fractions were prepared from strains bearing wild-type and N-terminally deleted Est2p and tested for telomerase primer extension activity using TEL66 as the primer oligonucleotide.

Loss of telomerase activity in the mutants cannot be accounted for by loss of protein expression-stability alone. To determine if reduced telomerase activity of the deletion and point mutants was due to reduced expression-stability of Est2p, we determined the amount of protein A-tagged Est2p in the mutant strains by Western analysis. To minimize potential variations introduced by IgG-Sepharose binding, we first directly analyzed unfractionated extracts for the presence of Est2p. An immunoreactive species of ~115 kDa can be detected in extracts from the tagged strain, but not from the untagged strain, supporting the specificity of our assay (Fig. 5A). As expected, the two functionally defective mutants that exhibited wild-type levels of in vitro telomerase activity (D66A and N104AV105A) had wild-type levels of Est2p (data not shown). Four mutants with reduced telomerase activity in vitro (G85A, Q138AF139A, G141A, and N-10) also exhibited levels of Est2p comparable to that of the wild-type strain (Fig. 5B). The 12-fold or greater loss of in vitro activity of these four mutants was therefore not due to reduced Est2p expression-stability. Interestingly, the four senescent mutants (W115A, F118AH119A, G123A, and N-30) did manifest a significant reduction in Est2p level such that it was not possible to detect these polypeptides unequivocally in unfractionated extracts (data not shown). However, following IgG-Sepharose purification, even these mutant proteins can be clearly visualized in Western analysis (Fig. 5C). The increased background in these latter assays (marked by a vertical bar to the right of the panel) came from IgG that was released by heating of the IgG-Sepharose beads in SDS and that reacted with the secondary antibody. Using signals derived from different amounts of Sepharose beads carrying wild-type Est2p as standards, these four mutant polypeptides appear to be present at approximately one-third to one-fifth of the wild-type protein level (compare lanes 3 to 6 with lane 1 and lane 2). The N-30 mutant polypeptide exhibited a slightly increased mobility by SDS-PAGE, further confirming the authenticity of our signal (Fig. 5C, lane 6). Given that telomerase activity was reduced by 50-fold or more in these senescent mutants, it appears that the mild reduction in Est2p level cannot solely account for the enzymatic defect.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Analysis of mutant protein expression. (A) Whole-cell extracts were prepared from strains whose Est2p was either tagged (+) or untagged () with the IgG binding domain of protein A and subjected to Western analysis. For each extract, 300 µg of total protein was examined. The position of the protein A-tagged Est2p is indicated by an arrow. (B) Est2p levels in whole-cell extracts from wild-type (W.T.) or mutant strains were analyzed by Western blotting. For each extract, 500 µg of total protein was examined. The position of the protein A-tagged Est2p is indicated by an arrow. (C) Est2p levels in whole-cell extracts from wild-type (W.T.) or mutant strains were assessed by affinity precipitation followed by Western analysis. For each extract, 4 mg of total proteins was incubated with 40 µl of IgG-Sepharose resin with gentle agitation at 4°C for 16 h. The beads were washed extensively, and the indicated amount of Sepharose was boiled in SDS-PAGE loading buffer. The eluted proteins were then subjected to immunoblotting. The position of the protein A-tagged Est2p is indicated by an arrow. The increased background (marked by a vertical bar) was due to IgG that eluted from the beads and that cross-reacted with the secondary antibody.

View larger version (54K): [in this window] [in a new window]   FIG. 6.   Several Est2p mutants can cause telomere shortening when overexpressed in the presence of wild-type protein. (A) W303 was transformed with a pYX212 plasmid expressing mutated Est2p. After restreaking of the transformants three times, chromosomal DNAs were isolated from the strains, digested with PstI, and analyzed for telomere lengths. DNAs were also isolated from untransformed W303 and tested for comparison. The source of the DNA is indicated at the top of the panel. (B) DNAs from strains overexpressing different Est2p mutant proteins were isolated and digested as for panel A and tested for telomere lengths. The source of the DNA is indicated at the top of the panel. The mobilities of several molecular size standards (in kilobases) are indicated at the sides.

Identification of a protease-resistant stable domain in the N-terminal region of Est2p. To begin to biochemically dissect the TERT polypeptide, we attempted to express recombinantly the N-terminal region of Est2p in Escherichia coli as MBP (maltose-binding protein) fusion proteins. To facilitate purification, the proteins were also fused to a six-His tag at its C terminus. Three fragments, 1-304, 1-270, and 1-160, were chosen for initial characterization as parts of the MBP fusion protein [designated MBP-Est(1-304)p, MBP-Est2(1-270)p, and MBP-Est2(1-160)p, respectively]. Interestingly, the two larger fragments appear to be sensitive to proteolysis in E. coli. For example, following affinity purification over a maltose column, fractions derived from the MBP-Est2(1-304)p-overproducing strain contained not only the full-length fusion polypeptide but also several smaller fragments (Fig. 7A, lane 1). These smaller fragments most likely resulted from proteolysis of the Est2p segment in vivo, because they still retained the MBP domain, and because the MBP domain on its own was stably expressed in E. coli (Fig. 7A, lane 6). Based on their size, the proteolyzed fragments appear to retain ~160 amino acids of Est2p (Fig. 7A, compare lanes 1 and 2). Interestingly, the MBP-Est2(1-160)p can be easily overproduced and purified as a single polypeptide from E. coli, again suggesting that this segment of Est2p, encompassing the N region and GQ motif, can form a stable domain in vivo that is resistant to proteolysis.

View larger version (25K): [in this window] [in a new window]   FIG. 7.   The Est2(1-160)p fragment exhibits a nonspecific nucleic acid binding activity. (A) Several MBP-Est2p fusion proteins were expressed and purified from E. coli using both nickel-affinity and maltose-affinity columns. The resulting preparations were analyzed in an SDS-10% polyacrylamide gel. The identities of the fusion proteins are indicated at the top of the panel. All fusion proteins (lanes 1 to 5) contain ~40 kDa of an MBP domain besides the Est2p fragments. The MBP (lane 6) derived from the original cloning vector contains an extra protein fragment beyond the polylinker and is therefore ~48 kDa in size. (B) Increasing amounts of MBP and MBP-Est2(1-160)p were incubated with 20 ng of in vitro-transcribed, labeled TLC1 RNA. The resulting mixtures were passed through a nitrocellulose filter, and the percentage of probe retained on the filter was plotted against the amount of protein used. Assays were performed in duplicate, and the averages and spreads are indicated. (C) Filter binding assays were performed using 1.3 µg of MBP-Est2(1-160)p and 20 ng of TLC1 RNA in the presence of increasing Mg concentrations. The percentage of probe retained on the filter was plotted against the Mg concentration. (D) Filter binding assays were performed using 1.3 µg of MBP-Est2(1-160)p and 20 ng of TLC1 RNA in the presence of increasing sodium acetate concentrations. The percentage of probe retained on the filter was plotted against the sodium acetate concentration (taking the amount retained in the presence of no salt as 100%). (E) Filter binding assays were performed using increasing amounts of MBP-Est2(1-160)p and 20 ng of either TLC1 RNA or antisense TLC1 RNA probe. The percentage of probe retained on the filter was plotted against the amount of protein used. (F) Filter binding assays were performed using 1.3 µg of MBP-Est2(1-160)p and 10 ng of labeled TLC1 RNA in the presence of increasing amounts of three different unlabeled DNA competitors: denatured salmon sperm DNA, double-stranded linear DNA, and single-stranded (SS) circular DNA. The percentage of probe retained on the filter was plotted against the ratio of competitor to probe (taking the amount retained in the presence of no competitor as 100%).

The N-terminal domain of Est2p possesses a nucleic acid binding activity. One potential function for the N-terminal domain is involvement in protein-RNA interactions. To test this idea, we assayed the N-terminal fusion protein for RNA binding activity using a filter retention assay. Initial studies employed MBP-Est2(1-160)p and ³²P-labeled full-length TLC1 RNA. As shown in Fig. 7B, the amount of RNA retained on the filter increased with increasing protein concentrations. At the highest protein concentration used (0.05 µg/µl), ~25% of the input RNA was retained on the filter. The apparent dissociation constant was about 5 µM. The amount of RNA retained by MBP was substantially less than that by the fusion protein, indicating that RNA binding was mediated by the N-terminal Est2p fragment.

The first 50 amino acid residues of Est2p are required for the nucleic acid binding activity. To further define the nucleic acid binding domain, we constructed a series of plasmids for expressing subfragments of the Est2p N-terminal domain. As before, these subfragments were fused to both an MBP and a six-His tag. The fusion proteins were expressed in and purified from E. coli as previously described (Fig. 7A) and used in filter binding assays.

View larger version (13K): [in this window] [in a new window]   FIG. 8.   The first 50 amino acid residues of Est2p are largely responsible for the nucleic acid binding activity exhibited by the N-GQ fragment. (A) Filter binding assays were performed using 1.3 µg of MBP, MBP-Est2(1-50)p, and MBP-Est2(1-160)p and 20 ng of labeled TLC1 RNA. The percentage of probe retained on the filter for each protein was plotted. (B) Filter binding assays were performed using increasing amounts of N-terminally truncated fusion proteins and 20 ng of labeled TLC1 RNA. The percentage of probe retained on the filter was plotted against the amount of protein used.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have investigated the function of the N-terminal region of Est2p (yeast TERT) by a combination of HMM-based sequence comparison, mutagenesis, and biochemical studies. In this report, we describe the identification of four phylogenetically conserved non-RT motifs (named GQ, CP, QFP, and T) located in the N-terminal region of TERTs. The locations of these motifs within Est2p are as follows: GQ, residues 45 to 163; CP, residues 245 to 265; QFP, residues 267 to 343; and T, residues 367 to 413. Alanine substitutions of conserved GQ motif residues confirmed the functional importance of this motif. Indeed, the identification of two phenotypic classes of mutations within this motif suggests that it may mediate at least two distinct biochemical functions. Finally, we showed that the extreme N-terminal, nonconserved region (N region) of TERT possesses a nonspecific nucleic acid binding activity when analyzed in isolation. Furthermore, the N region proved to be important for telomere maintenance and telomerase activity in the context of the native telomerase RNP.

Comparison of the unigenic evolution approach and the HMM-based alignment approach in identifying functionally important TERT regions. While our work was in progress, Friedman and Cech (11) reported the identification of essential yeast TERT N-terminal domains using a unigenic evolution approach. In this approach, the gene of interest is heavily mutagenized, and functional variants are selected. Essential and dispensable regions of the protein are then identified by statistically analyzing the distribution of missense and silent mutations. This analysis led to the identification of four essential regions in the Est2p N-terminal portion: region I, residues 31 to 163; region II, 214 to 265; region III, 285 to 374; and region IV, 378 to 432. A casual comparison between these regions and our motifs immediately indicates that they are largely concordant, with the GQ motif corresponding to region I, the CP motif corresponding to region II, and the QFP and T motifs corresponding to regions III and IV, respectively (Fig. 1B).

The function of the GQ motif. While mutagenesis indicates that the GQ motif mediates an important function(s), its precise mechanisms are not understood. As described earlier, two mutations in nonconserved residues failed to affect telomerase function in vitro and in vivo. In contrast, eight out of eight mutants with changes in conserved residues show defective telomerase function in vivo. These mutations can be further classified according to the level of telomerase primer extension activity in vitro: one class of mutants (called class A, six of eight mutants) has 1/12 or less the wild-type levels of activity, while the other class (called class B, D66A and N104AV105A) has nearly wild-type levels of activity. The identification of phenotypically distinct mutants suggests that the GQ motif may play at least two functions in telomere maintenance. Potential defects for the two classes of mutants are discussed separately below.

The function of the N region. Our results suggest that the first 50 amino acid residues of Est2p (N region) are required for telomere maintenance in vivo and telomerase activity in vitro. These 50 amino acid residues, when separated from the rest of the RNP, are also capable of mediating nucleic acid binding in vitro. Deleting the first 10 amino acids of Est2p results in significant telomere shortening and an ~50-fold reduction in telomerase activity. The same deletion reduced the binding activity of the 1-160 fragment by fivefold. Deleting the first 30 or 50 amino acids had more severe effects both on the function of the full-length protein and on the binding activity of the 1-160 fragment. The correlation between function of the full-length protein and the binding activity of the N-terminal fragment suggests that the binding activity reflects an important physiologic function. We have been unable to express and purify a significant amount of full-length Est2p. Thus, it is not possible to compare the binding properties of N-terminal domain with those of the full-length protein. However, given the localization of the RT motifs in the C terminus of Est2p, the N-terminal domain is unlikely to account completely for the binding activity of the full-length protein.

The N-GQ fragment may constitute a stable domain of TERT. Sequence analysis suggests that the conserved GQ motif is followed in the alignment by a presumed flexible spacer, which exhibits little conservation and is variable in length. This conjecture is supported by our expression studies with E. coli. As described in the Results section, while the 1-160 fragment can be easily overproduced and purified from E. coli, larger fragments are quite susceptible to proteolysis. In addition, unigenic evolution analysis indicates that this linker region may be largely dispensable for function. The sequence alignment, mutagenesis, and biochemical studies, taken together, suggest that all TERTs may have at least a bipartite domain organization, with conserved N-terminal (N-GQ fragment) and C-terminal (CP-QFP-T-RT fragment) domains connected through a flexible spacer. The identification of a stable domain of TERT should open the way toward detailed structural analysis of at least an important part of telomerase. Given that telomerase has been shown to be an attractive anticancer drug target, such a structural study will no doubt be interesting both from a biochemical and from a pharmacological perspective.