RNA binding domain of telomerase reverse transcriptase

Telomerase is a ribonucleoprotein reverse transcriptase that extends the en ds of chromosomes. The two telomerase subunits essential for catalysis in v itro are the telomerase reverse transcriptase (TERT) and the telomerase RNA , Using truncations and site-specific mutations, we identified sequence ele ments of TERT and telomerase RNA required for catalytic activity and protei n-RNA interaction for Tetrahymena thermophila telomerase. We found that the TERT amino and carboxyl termini, although evolutionarily poorly conserved, are nonetheless important for catalytic activity. In contrast, high-affini ty telomerase RNA binding requires only a small region in the amino terminu s of TERT, Surprisingly, the TERT region necessary and sufficient for telom erase RNA binding is completely separable from the reverse transcriptase mo tifs, The minimal Tetrahymena TERT RNA binding domain contains two sequence motifs with ciliate-specific conservation and one TERT motif with conserva tion across all species. With human TERT, we demonstrate that a similar reg ion within the TERT amino terminus is essential for human telomerase RNA bi nding as well. Finally, we defined the Tetrahymena telomerase RNA sequences that are essential for TERT interaction. We found that a four-nucleotide r egion 5' of the template is critical for TERT binding and that the 5' end o f telomerase RNA is sufficient for TERT binding. Our results reveal at leas t one evolutionarily conserved molecular mechanism by which the telomerase reverse transcriptase is functionally specialized for obligate use of an in ternal RNA template.