Introduction

Telomerase is a specialized DNA polymerase that extends the 3' ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B' position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes. ^[1]

TERT is just one subunit of the Telomerase protein complex. TERT acts in junction with the TEN complex and a protein known as dyskerin in a homodimer. Each of the two parts of the dimer contains one TERT, one TEN, and one dyskerin molecule. Shown here is one TERT protein.

Structural Overview

TERT is a specialized reverse transcriptase belonging to the same protein family as DNA Polymerase and as such it shares the same structural motifs with a slightly different arrangement. On the whole polymerases are composed of structurally conserved motifs: the "fingers" "thumb" and "palm". TERT polymerase is similar in structure to retroviral reverse transcriptases such as HIV Reverse transcriptase. However, unlike other transcriptases TERT exists as a stable RNA-Protein complex, with an RNA molecule acting as the template molecule for dNTP addition. It is this RNA primer which allows for the high fidelity and high processivity of TERT Polymerase.

The binding site of this protein consists of a

that is configured in such a way that the DNA is inserted with its 3' end at the active site for dNTP addition. The structure shown here is a TERT complex with a DNA-RNA hairpin loop hybrid. The hybrid in the crystal structure shown here was developed in such a way that it binds similarly to the enzyme's RNA template strand, since the exact telomeric repeat sequence is not known for T. Castaneum ^[1]

Major Domains

The four major domains of TERT Polymerase are as follows

Thumb

The thumb domain of the enzyme is implicated in DNA binding and processivity. , form hydrogen bonds with the phosphates in the DNA backbone and keep the base pairs oriented towards the inside of the binding pocket. A portion of the thumb domain, residues are also responsible for guiding the DNA substrate into the binding pocket through bulky interactions and electron repulsion. The thumb domain runs almost completely parallel to the curvature of the DNA substrate, further supporting the hypothesis that the thumb domain guides the DNA into place within the enzyme.^[1]

Palm

The palm domain contains the active site of the enzyme. Residing in this active site are (Asp 251, 343, and 344) which form a complex with the nearby Mg2+ ion and the DNA and incoming dNTPs.

The palm domain also contains the the section of the TERT Polymerase molecule where the incoming dNTPs reside before addition to the lengthening 3' end of the DNA strand. ^[1]

Finger

The finger domain in other polymerases, undergoes significant structural changes in order to bring the nucleotides present in the nucleotide binding pocket close enough to bind to the expanding DNA strand. However in polymerase the fingers seem to take a rigid closed ring conformation. This conformation seems to suggest that TERT Polymerase has a pre-formed active site, very similar in homology to that of Hepatitis C viral RNA polymerase.

This could, however, be a case where the protein was crystallized in the closed conformation. If this is the case, then it becomes clear that for the enzyme to be transitioned into the open conformation, a large amount of energy would be required. This energy could be supplied by certain accessory proteins similar to those found in conjunction with DNA Polymerases.^[1]

TRBD

The TERT RNA Binding Domain is the portion of the protein that contains the stable, complexed RNA template strand. It is here that the DNA-RNA hairpin loop in the structure binds.^[1]

Enzyme Activity

Repeat Addition Processivity

TERT Polymerase has the unique ability to add multiple repeat sequences of DNA to the end of chromosomes, a process which is known as Repeat Addition Processivity. This high amount of processivity can be attributed to the N-terminal association of the TERT subunit with the TEN subunit, the RNA templating region. In this structure unfortunately we do not see the actual association of RNA with the TRBD, however comparisons with other studies and known structural homologies we know that the RNA would bind in such a way as to place the 5' end of the template near the opening of the RNA binding pocket of the TRBD domain.

Because the RNA template has such a high affinity for the TERT molecule, it forces the enzyme to stall once dNTP addition has reached the 5' end of the RNA template. This causes TERT to dissociate from the expanding DNA chain and re-attach for another round of telomeric addition.

Conformational Changes

Comparisons between the substrate bound TERT molecule and the substrate free molecule show small rigid body conformational changes in the positions of the subunits of the enzyme. These small conformational changes translate to a 3.5 angstrom decrease in the diameter of the interior cavity of the closed ring. The precise role of this conformational change is not known, although it may have to do with RNA binding in vivo

Structural Similarity to HIV Reverse Transcriptase

(1hys).

It has been postulated that the mechanism by which TERT performs elongation of the telomeres is similar to the mechanism used by reverse transcriptases such as those found in retroviruses like HIV. Comparing the two proteins, it can be seen that there exist subtle differences in the structure, but the residues of the active sites are highly conserved across the two types of proteins, and the structural domains remain the same across the two as well. For example, the thumb loop of both is responsible for guiding the DNA substrate into the binding pocket of the enzyme, and orienting it in the correct direction. The Tyr256 and Gln308 of the nucleotide binding pocket are also found in HIV-RT. ^[1]

Current Research

Aging Research

Because the shortening of the telomere is directly responsible for the eventual senescence of the cell and by extension the aging of the individual, research on effectively elongating the telomere either by introducing synthetic TERT protein or by stimulating the natural production of TERT by the cells is a biological study of great significance.

In 2012 researchers at the Spanish National Cancer Research Centre (CNIO) showed a 24% increase in lifespan for mice who were injected with a virus containing the genes for telomerase production. Telomerase is actually expressed in most in vivo cells, however its levels are not high enough to completely combat the effects of the damage done by replication. With the addition of Telomerase genes by an adeno-associated Virus (AAV) late in the life of the mouse a longer lifespan was observed. Whereas previous studies have shown the possibility of altering telomerase genes during embryonic development to elongate the lifespan, this is the first study to show that an in vivo gene therapy can be effective in doing the same.

The results also showed no higher negligibly higher instances of cancer in these mice, as has been observed in several other studies wherein telomerase is reactivated. In addition to this, a slight rejuvenating effect was seen, with metabolism and mitochondrial fitness increasing a considerable amount. ^[2]

Cancer Research

Telomerase function has been heavily linked with cancer proliferation and as such it is one of the most common markers for cancer testing. Because of this heavy link to cancer, it is a prime target for many types of cancer research.^[3]

TERT Polymerase has been shown to be inhibited by the human protein PinX1. PinX1 works by binding to the site where the RNA template is usually bound. The PinX1 protein is usually found in the nucleolus and nucleoplasm in humans, but in cancer cells TERT is found in the nucleoplasm. It is postulated that PinX1 acts as a tumor suppressor for this reason. ^[4]