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J. Biol. Chem., Vol. 275, Issue 31, 24199-24207, August 4, 2000
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From the Howard Hughes Medical Institute, Department of Chemistry
and Biochemistry, University of Colorado,
Boulder, Colorado 80309-0215
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.
A Mutant of Tetrahymena Telomerase Reverse
Transcriptase with Increased Processivity*
*
This work was supported by a Human Frontier Science Program
Long-term Fellowship (to T.M.B) and the Howard Hughes Medical Institute.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Howard Hughes Medical
Institute, Dept. of Chemistry and Biochemistry, University of Colorado,
Boulder CO 80309-0215. Tel.: 303-492-8606; Fax: 303-492-6194; E-mail:
thomas.cech@colorado.edu.
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