A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity

The protein catalytic subunit of telomerase (TERT) is a reverse transcripta se (RT) that utilizes an internal RNA molecule as a template for the extens ion of chromosomal DNA ends. In all retroviral RTs there is a conserved tyr osine two amino acids preceding the catalytic aspartic acids in motif C, a motif that is critical for catalysis, In TERTs, however, this position is a leucine, valine, or phenylalanine, We developed and characterized a robust in vitro reconstitution system for Tetrahymena telomerase and tested the e ffects of amino acid substitutions on activity. Substitution of the retrovi ral-like tyrosine in motif C did not change overall enzymatic activity but increased processivity, This increase in processivity correlated with an in creased affinity for telomeric DNA primer. Substitution of an alanine did n ot increase processivity, while substitution of a phenylalanine had an inte rmediate effect. The data suggest that this amino acid is involved in inter actions with the primer in telomerase as in other RTs, and show that mutati ng an amino acid to that conserved in retroviral RTs makes telomerase more closely resemble these other RTs.