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A mutant of Tetrahymena telomerase rever ... ase with increased processivity
A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity.
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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.
Bryan TM, Goodrich KJ, Cech TR
The Journal of biological chemistry
2000-08-04 00:00
275
31
24199-207
Amino Acid Motifs,Animals,Models, Biological,Movement,Mutation,Telomerase,Tetrahymena thermophila,Telomerase
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA
J. Biol. Chem.
0021-9258
10.1074/jbc.M003246200
M003246200
130
True
10807925
