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Molecular and Cellular Biology, September 1999, p. 6207-6216, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Two Inactive Fragments of the Integral RNA Cooperate To Assemble Active Telomerase with the Human Protein Catalytic Subunit (hTERT) In Vitro

Valerie M. Tesmer, Lance P. Ford, Shawn E. Holt,dagger Bryan C. Frank, Xiaoming Yi, Dara L. Aisner, Michel Ouellette, Jerry W. Shay, and Woodring E. Wright*

Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039

Received 26 April 1999/Returned for modification 28 May 1999/Accepted 14 June 1999

We have mapped the 5' and 3' boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.

* Corresponding author. Mailing address: Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9039. Phone: (214) 648-2933. Fax: (214) 648-8694. E-mail: wright{at}utsw.swmed.edu.

dagger Present address: Department of Pathology and Human Genetics, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA 23298.

Molecular and Cellular Biology, September 1999, p. 6207-6216, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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