Telomerase Catalytic Subunit TERT in Tribolium castaneum

  Shirley Lu '15 and Nicole Valentini '14


I. Introduction

The telomerase catalytic subunit TERT in flour beetle, Tribolium castaneum, is a specialized ribonucleoprotein (RNP) reverse transcriptase responsible for extending the 3' ends of eukaryotic linear chromosomes, which helps genomic stability and cell viability. It binds to RNA templates and telomeric DNA. Lack of telomerase could lead to a reduction in cell size and cellular aging.

Telomerase is capable of adding multiple identical repeats of DNA. Initiation of telomere replication begins with the pairing of an RNA-templating region with an incoming single-stranded DNA primer. Completion of the telomere extension leads to the dissociation of the RNA-DNA hybrid. The DNA is then repositioned so that the DNA end is at the active site of TERT. The RNA-DNA pairing is at the other end of the template. Regulated by telomere binding proteins, telomerase is associated with the chromosome end until several telomeric repeats have been added.

II. General Structure

TERT is structurally similar to several other eukaryotic and prokaryotic binding proteins, namely RNA polymerases and B-family DNA polymerases. Like these proteins, it mimics the structure of a hand with finger, palm, and thumb domains essential for nucleotide associations and catalysis. The finger domain is involved in nucleotide binding and processivity , the palm domain contains the active site of the enzyme , and the thumb domain is involved in DNA binding and processivity . These domains are organized into a ring configuration as seen in a typical substrate free enzyme. The image presented represents a crystallized full length active T. castaneum TERT with an RNA-DNA hairpin containing both an RNA-templating region and a complementary telomeric DNA , joined together by an RNA-DNA linker.

TERT binding to the RNA-template is mediated by motifs 2 and B' of the palm domain, which localize adjacent to and above the active site of the enzyme. Protein contacts with the backbone of the RNA template position the bases within the active site of the enzyme for nucleotide binding, allowing for selectivity. In general, TERT DNA interactions are mediated by the thumb loop and helix, which provide the stability for functional telomerase elongation and facilitate the positioning of the 3'-end nucleotides near the primer-grip region (also called motif E). Furthermore, motif E positions the 3'-end hydroxyl of the DNA primer at the active site of the enzyme, facilitating nucleotide addition. Overall, interactions between TERT, the RNA-templating region, and the DNA primer extended in elongation are important for providing more insight into how telomeres are replicated.

The RNA component of T. castaneum was designed from previous information, as its exact composition is not known. The RNA-DNA hairpin contains three-nucleotide overhangs at the 5' end of the RNA template, in order to keep the enzyme in its catalytic state when co-crystallizing. MG2+ and non-hydrolyzable nucleotides were used to facilitate co-crystallization of the protein-nucleic acid assembly.

III. RNA Binding

The DNA substrate with TERT is usually associated with the RNA-templating region TERT, which is an active polymerase in the presence of nucleic acids. The RNA template makes contact with conserved motifs. TERT also makes contact with the RNA template, which positions the solvent-accessible bases adjacent to the active site of the enzyme, allowing nucleotide binding. The TERT-RNA association further positions the 5' end of the RNA template at the RNA binding pocket. This is where the template boundary element, which is upstream of the RNA-template, binds. The 3' end of the DNA substrate at the active site of the enzyme is placed at the 3' end of the DNA substrate, allowing nucleotide addition to be accessible.

Interactions with the RNA-templating region are facilitated by the fingers, palm, and thumb domains. The 5'-end of RNA is in close proximity to the active site of the enzyme. Specifically, the 2'-OH cytosine is close enough to engage in hydrogen bonding with the backbone carbonyls of Val197 of motif 2 and Gly309 of motif B', placing cytosine near the active site. The incoming nucleotide substrate is then ready to base pair with it. Contact with uracil and the protein are aided by the short aliphatic side chain of Pro311 and the ribose group. The 5'-end bases of the templating region are stabilized with five ribonucleotides that are attached to the incoming DNA primer. Water molecules limit the contact between this region and TERT.

IV. DNA Binding

Interaction between TERT and the DNA substrate are mediated with backbone interactions and the thumb loop and helix. The thumb helix makes contact with the phosphodiester backbone and ribose groups of the RNA-DNA hybrid, sitting in the minor groove. The thumb loop, which is a part of the thumb domain, sits parallel to the twist of the DNA primer, creating a network of backbone-and-solvent-mediated interactions. These interactions include the side chains of Lys416 and Asn432, both of which can hydrogen bind with the DNA backbone. The thumb domain's contact with DNA positions the nucleotides at the 3' end within the primer-grip region, which is formed by the backbone of residues Cys390 and Gly391 . This guides the 3'-end DNA nucleotides towards the active site of the enzyme. The TERT active site consists of three amino acid residues: Val342, Tyr256, and Gln308, which form parts of two short loops that are located on the palm subdomain. Residues Tyr256 and Val342 form hydrophobic pockets that are adjacent to three catalytic apartates, and accommodate the base of the nucleotide substrate. The TRBD domain forms a deep cavity on the surface of TERT that forms a gap allowing for nucleotide entry into the hole of the ring. This hole is important as it forms during TERT-TER assembly and it serves as the area for the placement of the RNA template in the interior of the ring.

When TERT is bound to nucleic acids, there is a small conformational change in the interior cavity of the ring. The diameter's size decreases, which could be related to TERT's association with TER (full-length RNA). More specifically, a change in the thumb domain and the TRBD toward the center of the ring creates a more narrow binding pocket than that of a substrate free TERT. Though not very clear, it is believed that this structural change is essential for the association of TERT with TER.

V. Telomere Replication

Repeat addition processivity is a process where telomerase adds multiple identical repeats of DNA to the ends of chromosome. This process is highly facilitated by the enzyme's integral RNA-templating region (TER). When telomere replication is initiated, pairing of the RNA-templating region with the incoming single stranded DNA primer occurs. Evidence suggests that the RNA template serves as the facilitator for several rounds of nucleotide addition and selectivity.

Telomerase is able to add multiple identical repeats to telomeres mainly by the association of the N-terminal portion of TERN with TER, the telomeric overhang, and the IFD motif. The TEN domain (which is not shown, but is indicated in pink in the link) is believed to be an important factor in addition processivity, as well as the association of TRBD with TER. Associations between TER and TRBD force the enzyme to stall when reaching the nucleotide located at the 5' end of this RNA template, halting replication. If this stalling occurs for long periods of time, the complex is destabilized and dissociation of the RNA-DNA heteroduplex results in the initiation of another round of telomere replication.


TERT-nucleic acid associations and domain organizations are similar to the structures observed for HIV RTs. In both systems, pairing of the templating region with the incoming DNA primer is necessary. Furthermore, the 3'-end of the DNA needs to be placed in the enzyme's active site in order for nucleotide addition to occur. Higher affinity binding also occurs in both structures due to domain rearrangements - the 3' end of DNA is more tightly bound to the active site during the enzymes' catalysis.
Both TERT and HIV RT involve the 2 and B' of the fingers and palm domains motif
. These positions assist the positioning of the solvent-accessible bases of the RNA template so that it is close to the active site for nucleotide binding. When either TERT or HIV RT interacts with DNA, the substrate is mediated by the thumb domain. Common mechanistic aspects of DNA replication, such as these, are seen in both substrates.

VII. References

Gillis, Andrew J., Anthony P. Schuller, and Emmanuel Skordalakes. 2008. Structure of the Tribolium castaneumi telomerase catalytic subunit TERT. Nature Structural and Molecular Biology 455: 633-637.

Mitchell, Meghan, Andrew Gillis, Mizuko Futahashi, Haruhiko Fujiwara, and Emmanuel Skordalakes. 2010. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nature Structural and Molecular Biology 17: 513-519.

Osanai, Mizuko, Kenji K. Kojima, Ryo Futahashi, Satoshi Yaguchi, and Haruhiko Fujiwara. 2006. Identification and characterization of the telomerase reverse transcriptase of Bombyx mori (silkworm) and Tribolium castaneum (flour beetle). Gene Section Evolutionary Genomics 376: 281-289.

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