Abstract
The protein catalytic subunit of telomerase (TERT) is a reverse transcriptase (RT) that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. In all retroviral RTs there is a conserved tyrosine 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 effects of amino acid substitutions on activity. Substitution of the retroviral-like tyrosine in motif C did not change overall enzymatic activity but increased processivity. This increase in processivity correlated with an increased affinity for telomeric DNA primer. Substitution of an alanine did not increase processivity, while substitution of a phenylalanine had an intermediate effect. The data suggest that this amino acid is involved in interactions with the primer in telomerase as in other RTs, and show that mutating an amino acid to that conserved in retroviral RTs makes telomerase more closely resemble these other RTs.
Highlights
Telomeres form a protective cap on chromosome ends and are usually composed of short G-rich DNA repeats complexed with proteins [1]
Reconstitution and Characterization of Telomerase Activity in Vitro—It has previously been demonstrated that Tetrahymena The protein catalytic subunit of telomerase (TERT) protein, when coexpressed with Tetrahymena telomerase RNA in rabbit reticulocyte lysates, forms a complex capable of telomerase activity [15] and that formation of this complex is dependent on factor(s) present in the reticulocyte lysate [22]
Activity was abolished by the use of either a telomerase RNA lacking the template domain or a TERT protein with the catalytic aspartate in motif A substituted by alanine, demonstrating that the activity observed is telomerase activity
Summary
HIV RT activity (22–30% of wt levels; Refs. 32 and 33), and had no effect on murine leukemia virus RT activity [34]. The tyrosine mutant (L813Y), while not showing any change in telomerase activity level, did show an increase in enzyme processivity. This is an unusual case where a single amino acid substitution provides an “improvement” in enzyme action. Because this substitution does not occur in any of the nine TERTs that have been identified, we propose that it may be advantageous to telomerases to synthesize rather small numbers of repeats in a single enzymatic cycle
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