Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the 7 proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labeling. Fitting with high-resolution structures reveals the organization of TERT, TER, and p65 in the RNP catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3′ exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.

Telomerase copies its internal RNA template to synthesize telomeric DNA repeats. Unlike other polymerases, telomerase can retain its single-stranded product through multiple rounds of template dissociation and repositioning to accomplish repeat addition processivity (RAP). Tetrahymena telomerase holoenzyme RAP depends on a subunit, Teb1, with autonomous DNA-binding activity. Sequence homology and domain modeling suggest that Teb1 is a paralog of RPA70C, the largest subunit of the single-stranded DNA-binding factor replication protein (RPA), but unlike RPA, Teb1 binds DNA with high specificity for telomeric repeats. To understand the structural basis and significance of telomeric-repeat DNA recognition by Teb1, we solved crystal structures of three proposed Teb1 DNA-binding domains and defined amino acids of each domain that contribute to DNA interaction. Our studies indicate that two central Teb1 DNAbinding oligonucleotide/oligosaccharide-binding-fold domains, Teb1A and Teb1B, achieve high affinity and selectivity of telomericrepeat recognition by principles similar to the telomere end-capping protein POT1 (protection of telomeres 1). An additional C-terminal Teb1 oligonucleotide/oligosaccharide-binding-fold domain, Teb1C, has features shared with the RPA70 C-terminal domain including a putative direct DNA-binding surface that is critical for high-RAP activity of reconstituted holoenzyme. The Teb1C zinc ribbon motif does not contribute to DNA binding but is nonetheless required for high-RAP activity, perhaps contributing to Teb1 physical association with the remainder of the holoenzyme. Our results suggest the biological model that high-affinity DNA binding by Teb1AB recruits holoenzyme to telomeres and subsequent Teb1C-DNA association traps product in a sliding-clamp-like manner that does not require high-affinity DNA binding for high stability of enzyme-product association.

The eukaryotic reverse transcriptase, telomerase, adds tandem telomeric repeats to chromosome ends to promote genome stability. The fully assembled telomerase holoenzyme contains a ribonucleoprotein (RNP) catalytic core and additional proteins that modulate the ability of the RNP catalytic core to elongate telomeres. Electron microscopy (EM) structures of Tetrahymena telomerase holoenzyme revealed a central location of the relatively uncharacterized p50 subunit. Here we have investigated the biochemical and structural basis for p50 function. We have shown that the p50-bound RNP catalytic core has a relatively low rate of tandem repeat synthesis but high processivity of repeat addition, indicative of high stability of enzyme-product interaction. The rate of tandem repeat synthesis is enhanced by p50-dependent recruitment of the holoenzyme single-stranded DNA binding subunit, Teb1. An N-terminal p50 domain is sufficient to stimulate tandem repeat synthesis and bridge the RNP catalytic core, Teb1, and the p75 subunit of the holoenzyme subcomplex p75/p19/p45. In cells, the N-terminal p50 domain assembles a complete holoenzyme that is functional for telomere maintenance, albeit at shortened telomere lengths. Also, in EM structures of holoenzymes, only the N-terminal domain of p50 is visible. Our findings provide new insights about subunit and domain interactions and functions within the Tetrahymena telomerase holoenzyme.

The eukaryotic reverse transcriptase telomerase copies its internal RNA template to synthesize telomeric DNA repeats at chromosome ends in balance with sequence loss during cell proliferation. Previous work has established several factors involved in telomerase recruitment to telomeres in yeast and mammalian cells; however, it remains unclear what determines the association of telomerase with telomeres in other organisms. Here we investigate the cell cycle dependence of telomere binding by each of the seven Tetrahymena thermophila telomerase holoenzyme proteins TERT, p65, Teb1, p50, p75, p45, and p19. We observed coordinate cell cycle-regulated recruitment and release of all of the subunits, including the telomeric-repeat DNA-binding subunit Teb1. Using domain truncation and mutagenesis approaches, we investigated which subunits govern the interaction of telomerase holoenzyme with telomeres. Our results show that Teb1 is critical for telomere interaction of other holoenzyme subunits and demonstrate that high-affinity Teb1 DNA-binding activity is necessary and sufficient for cell cycle-regulated telomere association. Overall, these and additional findings indicate that in the ciliate Tetrahymena, telomerase recruitment to telomeres requires direct binding to single-stranded DNA, unlike the indirect DNA recognition through telomere-bound proteins essential in yeast and mammalian cells.

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