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Molecular and Cellular Biology, March 2007, p. 2130-2143, Vol. 27, No. 6
0270-7306/07/$08.00+0     doi:10.1128/MCB.01826-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Triple Helix within a Pseudoknot Is a Conserved and Essential Element of Telomerase RNA{triangledown}

Kinneret Shefer,1 Yogev Brown,1 Valentin Gorkovoy,1 Tamar Nussbaum,1 Nikolai B. Ulyanov,2* and Yehuda Tzfati1*

Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram, 91904 Jerusalem, Israel,1 Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158-25172

Received 26 September 2006/ Returned for modification 9 November 2006/ Accepted 19 December 2006

Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A·U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C·C+ triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C·U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.

* Corresponding author. Mailing address for Nikolai B. Ulyanov: Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94158-2517. Phone: (415) 476-0707. Fax: (415) 502-8298. E-mail: ulyanov{at}picasso.ucsf.edu. Mailing address for Yehuda Tzfati: Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram, 91904 Jerusalem, Israel. Phone: 972-2-6584902. Fax: 972-2-6586975. E-mail: tzfati{at}cc.huji.ac.il.

{triangledown} Published ahead of print on 8 January 2007.

Molecular and Cellular Biology, March 2007, p. 2130-2143, Vol. 27, No. 6
0270-7306/07/$08.00+0     doi:10.1128/MCB.01826-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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