The loop of the Est2-interacting stem-loop is not required for association of Est2 with the telomerase RNP. (A) Sequences deleted in the tlc1-62 mutant are indicated. (B) Telomere length of a tlc1-62 strain (lanes 2 and 3) compared to that of a TLC1 strain (lane 1). (C) Coimmunoprecipitation of ProA-Est2 (top) and HA₃-Est1 (bottom) with either TLC1 or tlc1-62 in parallel with tlc1-Δ148-440.

Chimeric TLC1 RNAs are functional in S. cerevisiae. (A) Diagram of three chimeric TLC1 molecules, constructed by replacing sequences in the S. cerevisiae TLC1 RNA with corresponding sequences from the S. kluyveri RNA. (B) Telomere length of a tlc1-Δ strain transformed with S. cerevisiae TLC1 (lanes 1 and 8), tlc1-76 (lanes 2 and 3), tlc1-91 (lanes 4 and 5), or tlc1-129 (lanes 6 and 7). (C) Coimmunoprecipitation of tlc1-76 and tlc1-91 with ProA-Est2 or HA₃-Est1 in parallel with tlc1-Δ148-440.

Regions adjacent to the stem-loop contribute to the interaction of the Est2 protein with TLC1. (A) Construction of a panel of deletion mutations on either side of the Est2-interacting stem-loop. Nucleotides that were deleted for each allele are indicated. (B) Telomere lengths of tlc1 mutants shown in panel A. Plasmids were transformed into a tlc1-Δ rad52-Δ strain and grown for ≈60 generations prior to analysis; data for tlc1-47 and tlc1-59 are shown in Fig. 3C. (C) Efficiency of coimmunoprecipitation of each tlc1 allele shown in part A with either ProA-Est2 or HA₃-Est1; the relative efficiency, normalized to tlc1-Δ148-440, was determined from three independent experiments.

O. nova and human pseudoknots can replace Est2-interacting sequences of the S. cerevisiae RNA. (A) Diagram of two chimeric TLC1 molecules (tlc1-94 and tlc1-96), constructed by replacing nucleotides 717 to 812 in the S. cerevisiae TLC1 RNA with the pseudoknot from the O. nova telomerase RNA (nucleotides 59 to 98) or a minimal version of the pseudoknot from the human telomerase RNA (nucleotides 90 to 183), respectively. (B) Growth after ≈75 generations of a tlc1-Δ strain harboring plasmids expressing TLC1, tlc1-94, tlc1-96, or vector. (C) Telomere length of tlc1 chimeras shown in part A. Plasmids were transformed into a tlc1-Δ strain and grown for ≈60 generations prior to analysis.

TLC1 genes from S. bayanus, S. castellii, and S. kluyveri are partially functional in S. cerevisiae. (A) Telomere length of a tlc1-Δ strain transformed with S. cerevisiae TLC1 (lanes 1 and 7), vector (lane 2), S. castellii TLC1 (lanes 3 and 4), or S. bayanus TLC1 (lanes 5 and 6). Telomere length was examined after ≈75 to 100 generations of growth, at a time when telomeres in the tlc1-Δ strain (lane 2) had begun to undergo rearrangements indicative of a recombination-dependent pathway for telomere maintenance. (B) Growth of a tlc1-Δstrain transformed with either vector or plasmids encoding the S. cerevisiae, S. bayanus, S. castellii, or S. kluyveri TLC1 gene. (C) Growth of a tlc1-Δ strain transformed with plasmids encoding S. kluyveri TLC1, S. cerevisiae TLC1, or vector in the presence or absence of a CDC13-EST1 fusion plasmid.

Defining a region of TLC1 required for interaction with Est2. (A) Sequences deleted in tlc1-47 and tlc1-59 are indicated by boxes. (B) Growth after ≈75 generations of a tlc1-Δ strain harboring plasmids expressing TLC1, tlc1-47, tlc1-59, or vector, with or without an ADH-EST2 plasmid (pVL369). (C) Telomere length of a tlc1-Δ strain transformed with TLC1 (lane 1), vector (lanes 2 and 7), tlc1-59 (lanes 3 and 4), or tlc1-47 (lanes 5 and 6). (D) Coimmunoprecipitation of TLC1 RNAs with either ProA-Est2 (top) or HA₃-Est1 (bottom). The tlc1-59 and tlc1-47 mutant RNAs as well as the control tlc1-Δ148-440 RNA were expressed on CEN plasmids that were introduced into a tlc1-Δ strain. A representative experiment is shown, as are the averaged values of three independent experiments.

The Est2 protein interacts with a stem-loop structure. (A) Diagram of the nucleotide changes introduced into the duplex portion of the stem: tlc1-29 and tlc1-30 (predicted to disrupt base pairing in a portion of the stem), tlc1-31 (which should restore base pairing as the result of compensating sets of mutations), and tlc1-34 (predicted to disrupt base pairing of the entire stem). A second set of mutations introduced into this stem are shown in the supplemental material (Fig. S2). (B) Telomere length of a tlc1-Δ rad52-Δ strain transformed with plasmids encoding wild-type TLC1 (lanes 1 and 8), tlc1-29 (lanes 2 and 3), tlc1-30 (lanes 4 and 5), and tlc1-31 (lanes 6 and 7) and grown for ≈60 generations. (C) Northern blot of tlc1-29, tlc1-30, and tlc1-31 after immunoprecipitation of ProA-Est2 or HA₃-Est1 from a tlc1-Δ strain harboring a tlc1-Δ148-440 plasmid. (D) Growth of a tlc1-Δ strain harboring either vector or plasmids that express TLC1, tlc1-29, tlc1-30, or tlc1-34.

Two previously identified stem-loops are conserved among the budding yeast telomerase RNAs. Predicted secondary structures for the Est1-interacting (A) and Est2-interacting (B) structures of the telomerase RNAs from six Saccharomyces species and from K. lactis. Determination of structures was based on primary sequence alignments and comparison to previously determined structural data (48, 56), aided by RNA Mfold version 3.1. The duplex portion of the Est1-interacting structure is extended by an additional 6 bp relative to that established previously (48), although this extension has not been experimentally tested. The primary sequence alignments that support the proposed structures shown in panel B are presented in the supplemental material (Fig. S1A). The regions highlighted in grey in the S. cerevisiae and K. lactis RNAs (B) indicate sequences capable of forming a potential pseudoknot (56).

Two alternative models for an additional Est2-interacting structure. (A) Proposed second stem-loop structure, shown for six Saccharomyces and six Kluyveromyces RNAs; the larger stem-loop corresponds to that depicted in Fig. 2B. In order to identify base pairs that covary, thetwo sets of six RNAs were coaligned on a block of conserved residues, which is boxed. Arrows indicate base pairs that covary among the Saccharomyces homologues compared to analogous base pairs in the S. cerevisiae RNA (top) and among the Kluyveromyces homologues compared to the K. lactis RNA (bottom). Arrowheads alongside the S. cerevisiae and K. lactis RNAs summarize the number of covarying base pairs identified within each set of six RNAs. The sequence alignments that support these proposed structures are shown in supplemental Fig. S1. (B) A pseudoknot structure that has been previously proposed for the K. lactis telomerase RNA (56) and which may also form in the S. cerevisiae RNA; the regions that base-pair to form the proposed pseudoknot are also highlighted in grey in Fig. 2B. (C) Diagram of the nucleotide changes introduced into the duplex portion of the proposed second stem-loop (see panel A). The position of the tlc1-111 deletion mutation, which removes one strand of the predicted duplex stem, is also indicated. (D) Telomere blot of strains bearing plasmids containing the tlc1-65, tlc1-66, tlc1-67, tlc1-71, tlc1-72, and tlc1-73 mutant RNAs. Note that predicted compensatory mutations (tlc1-67 and tlc1-73), which should restore base pairing of the predicted duplex structure, do not restore the telomere length defect.