Functional reconstitution of wild-type and mutant Tetrahymena telomerase

Autexier, C., Greider, C. W. (March 1994) Functional reconstitution of wild-type and mutant Tetrahymena telomerase. Genes Dev, 8 (5). pp. 563-75. ISSN 0890-9369 (Print)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/7523243
DOI: 10.1101/gad.8.5.563

Abstract

Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)n to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.

Item Type: Paper
Uncontrolled Keywords: Animals Base Sequence DNA Nucleotidylexotransferase/genetics/ metabolism Molecular Sequence Data Mutagenesis, Site-Directed Mutation RNA/genetics Repetitive Sequences, Nucleic Acid Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Telomere Templates, Genetic Tetrahymena/ enzymology
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > telomerase
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > telomeres
CSHL Authors:
Communities: CSHL labs > Greider lab
Depositing User: Matt Covey
Date: 1 March 1994
Date Deposited: 27 Aug 2015 21:13
Last Modified: 03 Nov 2017 20:27
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31400

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