Telomeres are the physical ends of eukaryotic chromosomes and are comprised of DNA repeat sequences. Telomeric DNA consists of thousands of repeats of a G-rich sequence at the 3í end of the chromosome terminus (Theimer et al., 2005). As DNA is replicated, the ends of the telomeres are degraded. For this reason telomeres are crucial in ensuring that the entire DNA sequence is replicated with out loss of genetic information due to chromosome degradation or rearrangement. Telomeres are maintained by telomerases which are ribonucleoproteins.

Human telomerase contains an RNA component (hTR) which is 451 nucleotides in length. Within the telomerase is the RNA template required for the reverse transcription of telomeres. The species dependent DNA tandem repeat sequences are added without the use of an external template. In this way, telomeric DNA synthesis functions similarly to retroviral reverse transcription. hTERT is the catalytic protein component of telomerase that is responsible for the reverse transcriptase activity. It has at least two distinct hTR binding regions, one of which is the pseudoknot (core) (Theimer et al., 2005). The core consists of the aforementioned template as well as a template boundary region and the helical region which folds into the pseudoknot (Theimer et al., 2005).

Nucleotides 93-184 comprise this pseudoknot region. Stem 1 is highlighted in red . Loop 1 is highlighted in yellow. This uridine rich loop lies in the major groove of stem 2 . Stem 2 is highlighted in blue . Loop 2 is highlighted in green . This adenine-rich loop lies in the minor groove of stem 1 .

The junction of both sets of stems and loops is where the essential tertiary interactions take place.

STRUCTURAL FEATURES

The stability of the pseudoknot is attributed to a network of tertiary structural interactions.

The single nucleotide bulge at U177 enables an alternative hairpin conformation which the wt pseudoknot structure is in equillibrium with. The mutant lacking the U177 bulge stabilizes the pseudoknot structure. For this reason a mutant construct was used for the experimentation as opposed to wild type pseudoknot structure (Theimer et al., 2005).

The pseudoknot structure's helical junction is stabilized by three major groove triplets and two minor groove triplets on either side of it .

U99-U102 of the uridine-rich loop 1 are completely conserved among vertebrates. A Hoogsteen A173-U99 base pair is formed at the junction between stem 1 and stem 2 .

This Hoogsteen interaction prevents coaxial stacking of stem 1 and stem 2.
This allows for the minor groove of stem 1 and the major groove of stem 2 to form a contiguous groove along one side of the pseudoknot containing both loops.

The three major groove base pair triples are comprised of Watson-Crick base parings between A and U in stem 2 and a Hoogsteen base pair with a U in loop 1 .

U100-U102 form base pair triples. U100 Hoogsteen base pairs with U115-A174 .

The hydrogen bonds are highlighted here .

U101 Hoogsteen base pairs with U114-A175. Similarly, U102 Hoogsteen base pairs with U113-A176. These three base pair triples form a triplex structure below the helical junction .

Two base triples lie above the helical junction. A171 hydrogen bonds with A117-U97 .

A172 likewise Hoogsteen base pairs with C116-G98 . Note the stacking of A171 over A172.

The high frequency of the base triples are, so far, unique among pseudoknot structures (Theimer et al., 2005). Experiments testing the function of these sets of base triples surrounding the junction by mutating them determined these base pairs essential in maintaining the pseudoknot structure's thermodynamic stability .

CONCLUSIONS

Telomerase activity depends on the tertiary structure stability of the pseudoknot. The pseudoknot structure of telomerase RNA is important is binding TERT in vivo in yeast and ciliates (Lin et al., 2004). However the exact location of TERT binding in vertebrates has not yet been determined (Theimer et al., 2005). Therefore, the pseudoknot region of the hTR which stabilizes the pseudoknot conformation, consisting of two stems and two loops, is highly conserved.

References

Theimer, C.A., Blois, C.A., Feigon, J. (2005). Structure of the Human Telomerase RNA Pseudoknot Reveals Conserved Tertiary Interactions Essential for Function. Mol. Cell 17, 617-682.
Lin, J.L., Ly, H., Hussein, A., Abraham, M., Pearl, S., Tzfati, Y., Parslow, T.G., Blackburn, E.H. (2004). A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein. PNAS 101, 14713-14718.
Voet, D., Voet, J. Biochemistry, 3rd edition. John Wiley and Sons, Inc., New Jersey: 2004, 1170.