Book of Abstracts: Albany 2003
June 17-21 2003
Biophysical Studies of Telomerase RNA Structure and Mutations Linked to Disease
Telomerase is a ribonucleoprotein responsible for the maintenance of telomeres, the physical ends of chromosomes. The RNA component of the protein contains the template for replication of the DNA as well as several conserved domains whose functions are only beginning to be elucidated. We have been investigating the structure, dynamics, and thermal stability of domains of human telomerase RNA (hTR). In two of these domains, called pseudoknot and CR7 domains, mutations have been found which are linked to autosomal dominant inheritance of the disease dyskeratosis congenita. Dyskeratosis congenita is a progressive bone marrow failure syndrome characterized by abnormal skin pigmentation, leukoplakia, and nail dystrophy. Autosomal dominant dyskeratosis congenita (DKC), as well as aplastic anemia, have been linked to mutations in the RNA component of telomerase. We have examined the effect of the DKC mutations on the structure and stability of human telomerase RNA pseudoknot and CR7 domains, using NMR and thermal melting.
The CR7 domain point mutation decreases stability and alters a conserved secondary structure thought to be involved in hTR accumulation in vivo. We find that pseudoknot constructs containing the conserved elements of the pseudoknot domain are in equilibrium with a hairpin conformation. The solution structure of the wild type hairpin reveals that it forms a continuous helix containing a novel run of three consecutive U·U and one U·C base pairs closed by pentaloops. A search of the RNA structure databases revealed no incidences of more than two consecutive U·U base pairs. Furthermore, the 6 base pairs unique to the hairpin conformation are phylogenetically conserved in mammals, suggesting that this conformation is functionally important. The DKC mutation in the pseudoknot domain results in a shift in equilibrium toward the hairpin form, primarily due to destabilization of the mutant DKC pseudoknot.
Our results provide insight into the effect of dyskeratosis congenita mutations on telomerase structure and suggest that the catalytic cycle of telomerase involves a delicate interplay between RNA conformational states, alteration of which leads to the disease state.
Chemistry and Biochemistry