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
). 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. ). 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. B).
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 (more ...) 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::Kanr 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).
A total of eight alanine substitutions at conserved GQ motif residues were constructed and tested: D66A, G85A, N104AV105A, W115A, F118AH119A, G123A, Q138AF139A, and G141A. Two additional mutants with substitutions in nonconserved residues (D93A and E154A) were also made and tested for comparison. As summarized in Table , three of these mutants (W115A, F118AH119A, and G123A), located quite close to one another, exhibited the most pronounced growth defects, giving rise to small and variably sized colonies suggestive of senescence (31
). The senescent phenotype, characterized by progressively slower cell multiplication, has been observed in several other telomerase knockout strains (25
). The display of senescence suggests that the W115A, F118AH119A, and G123A mutants are quite compromised in telomerase function. In contrast, the other mutants either did not show any growth defects or grew only slightly slower (compared to a strain carrying a plasmid containing the wild-type EST2
gene) and failed to exhibit signs of senescence upon repeated restreaking.
TABLE 1 Summary of in vivo and in vitro phenotypes of point mutants in the GQmotif
Chromosomal DNA was isolated from individual transformants following two restreaks (~50 generations) and assayed for telomere lengths. As shown in Fig. , the Y′ class of telomeres in all mutants with substitutions in conserved residues shows dramatic telomere shortening of at least 150 to 200 bp (all mutants except D93A and E154A, lanes 2 to 5, 8 and 9, and 12 to 21). The senescent mutants have even shorter telomeres and gave especially weak signals for hybridization (W115A, F118AH119A, and G123A, lanes 12 to 17). Thus, there is a good correlation between growth defects and telomere repeat loss, consistent with earlier findings. In contrast to mutations in conserved residues, the two strains with mutations in nonconserved residues (D93A and E154A) failed to show any evidence of telomere shortening.
FIG. 2 Telomere length determination in strains that contain mutations in the conserved and nonconserved residues within the GQ motif. The Δest2 strain that had been grown for ~25 to 50 generations was transformed with plasmids bearing either (more ...) 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
]), 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. A, 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. A).
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 (more ...)
Each mutant telomerase was tested side by side with the wild-type fraction using equal amounts of total protein (Fig. B). As expected, the control mutants that exhibited no telomere shortening had in vitro activities that were comparable to those of the wild-type enzyme (D93A and E154A, lanes 5 and 15). This result further demonstrates the reproducibility of our protocol. Six of the eight mutants with substitutions in conserved residues exhibited greatly reduced telomerase activity, ranging from ~12-fold reduction for G85A (lane 3) to more than 50-fold reduction for the senescent mutants (W115A and G123A, lanes 9 and 10). Interestingly, two mutants, D66A and N104AV105A (lanes 2 and 7), appear to uncouple telomerase activity in vitro from telomere maintenance in vivo; though the telomeres in these two strains are greatly shortened, the mutant enzymes exhibited nearly wild-type levels of activity. Other strains that had similar telomere length defects such as G85 and Q138AF139A (lanes 3 and 12) suffered a ~12- to 20-fold reduction in telomerase activity (Table ). All mutant fractions were assayed at least twice, and where a significant reduction in activity relative to that of the wild-type fraction was observed, the mutant activity as a percentage of the wild-type activity varied by less than 5%.
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.
Four deletion mutants missing amino acids 2 to 10, 2 to 20, 2 to 30, and 2 to 50 (abbreviated as N-10, N-20, N-30, and N-50, respectively) were tested in this system. As shown in Fig. A and Table , the N-10 and N-20 mutants exhibited significantly reduced growth rates in minimal medium on agar plates. However, they showed no evidence of senescence upon repeated restreaking (data not shown). In contrast, the N-30 and N-50 mutants gave rise to heterogeneously sized colonies suggestive of senescence. Chromosomal DNA from two independent clones of each strain was isolated following two restreaks and analyzed for telomere length by Southern hybridization. Consistent with the growth defects, all deletion mutants, including the smallest one (N-10), possessed significantly shortened telomeres relative to the control strain (Fig. B, compare lanes 1 and 8 with lanes 2 to 7). Both Y′-type telomeres (marked by a vertical bar) and X-type telomeres (marked by arrows) in the mutant strains were shortened, consistent with a general defect in telomere maintenance.
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 (more ...)
TABLE 2 Summary of in vivo and in vitro phenotypes of N-terminal deletions ofEst2p
The deletion mutants were also tested for defects in telomerase activity using the primer extension assay. As shown in Fig. C, fractions derived from the N-10 and N-20 strains exhibited significantly reduced telomerase activity compared to those from the control strain. Quantification by PhosphorImager analysis indicates that the reduction is approximately 40- to 50-fold. The N-30 and N-50 strains were not tested for in vitro activity. However, based on the growth defects, it is likely that these latter strains would have exhibited the same or less telomerase activity. We conclude that the N region of Est2p is required for full telomerase primer extension activity as measured in vitro.
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. A). 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. B). 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. C). 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. C, 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.
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 (more ...)
If the senescent mutant proteins were indeed defective in function (as opposed to defective only in expression-stability), then they might act in a dominant-negative fashion when overexpressed in the presence of wild-type protein. This was found to be indeed the case. Four mutants that caused senescence (W115A, F118AH119A, G123A, and N-30) were placed downstream of a strong constitutive promoter, and the resulting plasmids were introduced into a wild-type strain (W303). Following three restreaks (~75 generations), chromosomal DNAs were isolated from the transformants and analyzed for telomere length alteration (Fig. ). Consistent with a defect in function, all four mutants caused significant telomere shortening in the host strain (Fig. A, compare lane 3 with lanes 1 and 4; Fig. B, compare lanes 3 to 5 with lane 1). As expected, two mutant proteins that supported normal telomere maintenance (D93A and E154A) had no effect when overproduced (Fig. B, lanes 2 and 6), and an Est2p with an RT active site mutation (D670A) caused the most severe telomere shortening (Fig. A, lane 2). The other nonsenescent GQ motif mutants caused at most a slight shortening of telomeres (~50 bp) when overexpressed in W303, possibly because they retain a significant level of function (data not shown).
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 (more ...) 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. A, 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. A, lane 6). Based on their size, the proteolyzed fragments appear to retain ~160 amino acids of Est2p (Fig. A, 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.
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 (more ...) 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. B, 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.
We investigated several reaction parameters and found that both salt and Mg2+ concentrations significantly affected the efficiency of binding. RNA retention as a function of Mg2+ concentration is a bell-shaped curve, with a peak at ~5 to 10 mM (Fig. C). The protein-RNA interaction was favored at low salt concentrations; the binding efficiency was reduced by ~75% at 250 mM sodium acetate relative to no salt (Fig. D).
To determine the sequence specificity of binding, we generated both sense and antisense TLC1 RNA probes by in vitro transcription and compared their abilities to interact with the fusion protein. As shown in Fig. E, the extent of binding is only twofold higher for the sense probe, suggesting that the N-terminal domain, at least by itself, does not recognize RNA with significant sequence specificity. We also tested the effect of RNA length on the efficiency of binding using TLC1 RNA missing increasing numbers of 3′-end residues. The results indicate that the fusion protein has similar affinities for RNAs ranging from 450 to 1,300 nucleotides long (data not shown).
We next tested binding of the fusion protein to DNAs of different structure using a competition filter-binding assay. A fixed amount of fusion protein [MBP-Est2(1–160)p, 1.3 μg] was mixed with both a fixed amount of labeled TLC1 RNA (10 ng) and variable amounts of unlabeled DNA competitors. The resulting mixture was then subjected to filtration through a nitrocellulose membrane as previously described. Single-stranded circular
X174 virion DNA, sheared and denatured salmon sperm DNA, and double-stranded linear DNA were used as the competitors. As shown in Fig. F, at an RNA/DNA ratio of 2:1, both the
X174 DNA and denatured salmon sperm DNA reduced the binding efficiency by 60%. Comparable inhibition of RNA binding by double-stranded linear DNA was achieved at a ratio of ~8:1. We also tested short single-stranded telomere oligonucleotides (the G-rich strand) in the competition assay, and the same amount of these oligonucleotides (by weight) was no more effective than the other single-stranded DNAs in reducing the binding signal (data not shown). Thus, the Est2p(1–160) fragment can also bind DNA in a non-sequence-specific fashion, with a slight preference for single-stranded DNA over double-stranded DNA.
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. A) and used in filter binding assays.
As shown in Fig. A, a large deletion from the C-terminal end of the Est2p fragment had little effect on RNA binding. Even a fusion protein with only the first 50 amino acid residues of Est2p [MBP-Est2(1–50)p] showed binding to RNA comparable to that of MBP-Est2(1–160)p. In contrast, a 10-amino-acid deletion from the N terminus significantly reduced the RNA binding activity, by approximately fivefold. When 30 or 50 amino acids were deleted from the N terminus, the RNA binding activity was reduced to the background level (Fig. B; also see the MBP plot in Fig. B). A similar series of assays using a double-stranded linear DNA as the probe gave essentially identical results (data not shown). These observations suggest that the N region (first 50 amino acids) of Est2p is largely responsible for the nonspecific nucleic acid binding activity exhibited by the 1–160 fragment.
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 (more ...)