Telomeres are the physical ends of eukaryotic chromosomes and are comprised of a DNA repeat sequence (TTAGGG). Telomeric DNA consists of thousands of copies of this G-rich repeat at the 3’ terminal end of the chromosome. (Theimer et al., 2005). Over successive rounds of DNA replication, the chromosome ends fail to get fully replicated and so are slowly degraded over time. Telomeres capping these ends protect the genetic information in the chromosomes because only non-coding telomeric DNA is lost through rounds of replication. This ensures that the entire DNA sequence is replicated without loss of genetic information due to chromosomal degradation or rearrangement.

Telomeres are maintained by a ribonucleoprotein complex called telomerase. Human telomerase contains a reverse transcriptase-like protein component (hTERT) and an RNA component (hTR) which is 451 nucleotides in length. The template sequence for the telomeric repeat is carried on the integral RNA so there is no requirement for an external template for the reverse transcriptase component of telomerase. The RNA does more than just provide the template sequence; however, it plays an as yet unknown role in the catalytic activity as well. Within hTR there are at least two distinct hTERT binding regions, one of which is the core domain (containing the pseudoknot), the other is the CR4/CR5 domain (Theimer et al., 2005). The core domain contains the aforementioned template sequence, 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 . Stem 2 is highlighted in blue. Loop 1 is highlighted in yellow. This uridine rich loop lies in the major groove of stem 2 . Loop 2 is highlighted in green. This adenine-rich loop lies in the minor groove of stem 1.

The Hoogsteen base pair (A173-U99) at the stem/loop junction positions the stems such that the major and minor grooves are contiguous along the entire length of the structure.

STRUCTURAL FEATURES

The stability of the pseudoknot is attributed to a network of tertiary structural interactions centered at the stem/loop junction.

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.

These interactions are complemented by the Hoogsteen base pair at the junction.

The three major groove base pair triples are comprised of Watson-Crick base parings between an A and a U in stem 2 and a Hoogsteen interaction with a U in loop 1. U100-U102 form base pair triples. U100 interacts 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 .

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.