Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit

Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5'-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5'-end of the RNA template. The upstream DNA-TBE interaction may also function as an anchor for the subsequent realignment of the 3'-end of the DNA with the 3'-end of the template to enable initiation of synthesis of a new telomeric repeat.     

Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding

Telomerase, a ribonucleoprotein complex, replicates the linear ends of eukaryotic chromosomes, thus taking care of the "end of replication problem." TERT contains an essential and universally conserved domain (TRBD) that makes extensive contacts with the RNA (TER) component of the holoenzyme, and this interaction is thought to facilitate TERT/TER assembly and repeat-addition processivity. Here, we present a high-resolution structure of TRBD from Tetrahymena thermophila. The nearly all-helical structure comprises a nucleic acid-binding fold suitable for TER binding. An extended pocket on the surface of the protein, formed by two conserved motifs (CP and T motifs) comprises TRBD's RNA-binding pocket. The width and the chemical nature of this pocket suggest that it binds both single- and double-stranded RNA, possibly stem I, and the template boundary element (TBE). Moreover, the structure provides clues into the role of this domain in TERT/TER stabilization and telomerase repeat-addition processivity.