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Molecular and Cellular Biology, March 2001, p. 1888-1897, Vol. 21, No. 5
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.5.1888-1897.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions

François Bachand and Chantal Autexier*

Department of Anatomy & Cell Biology, McGill University, Montréal, Québec, Canada H3A 2B2, and Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec, Canada H3T 1E2

Received 14 September 2000/Returned for modification 16 October 2000/Accepted 28 November 2000

Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.

* Corresponding author. Mailing address: Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste. Catherine Rd., Montréal, Québec, Canada H3T 1E2. Phone: (514) 340-8260. Fax: (514) 340-8295. E-mail: cautex{at}po-box.mcgill.ca.

Molecular and Cellular Biology, March 2001, p. 1888-1897, Vol. 21, No. 5
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.5.1888-1897.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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