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Yehuda Tzfati

Publications and source records attributed to Yehuda Tzfati.

6 recordsLinked to original sources
PubMed2007

A triple helix within a pseudoknot is a conserved and essential element of telomerase RNA.

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.

Base Sequence↗
PubMed2004

A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein.

Telomerase synthesizes telomeric DNA by copying a short template sequence within its telomerase RNA component. We delineated nucleotides and base-pairings within a previously mapped central domain of the Saccharomyces cerevisiae telomerase RNA (TLC1) that are important for telomerase function and for binding to the telomerase catalytic protein Est2p. Phylogenetic comparison of telomerase RNA sequences from several budding yeasts revealed a core structure common to Saccharomyces and Kluyveromyces yeast species. We show that in this structure three conserved sequences interact to provide a binding site for Est2p positioned near the template. These results, combined with previous studies on telomerase RNAs from other budding yeasts, vertebrates, and ciliates, define a minimal universal core for telomerase RNAs.

Base Sequence↗
PubMed2003

A novel pseudoknot element is essential for the action of a yeast telomerase.

Telomerase contains an essential RNA, which includes the template sequence copied by the reverse transcription action of telomerase into telomeric DNA. Using phylogenetic comparison, we identified seven conserved sequences in telomerase RNAs from Kluyveromyces budding yeasts. We show that two of these sequences, CS3 and CS4, are essential for normal telomerase function and can base-pair to form a putative long-range pseudoknot. Disrupting this base-pairing was deleterious to cell growth, telomere maintenance, and telomerase activity. Restoration of the base-pairing potential alleviated these phenotypes. Mutating this pseudoknot caused a novel mode of shifting of the boundaries of the RNA template sequence copied by telomerase. A phylogenetically derived model of yeast TER structure indicates that these RNAs can form two alternative predicted core conformations of similar stability: one brings the CS3/CS4 pseudoknot spatially close to the template; in the other, CS3 and CS4 move apart and the conformation of the template is altered. We propose that such disruption of the pseudoknot, and potentially the predicted telomerase RNA conformation, affects polymerization to cause the observed shifts in template usage.

Base Pairing↗
PubMed2003

A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity.

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.

Base Sequence↗
PubMed2002

A bulged stem tethers Est1p to telomerase RNA in budding yeast.

It is well established that the template for telomeric DNA synthesis is provided by the RNA subunit of telomerase; however, the additional functions provided by most of the rest of the RNA (>1000 nucleotides in budding yeast) are largely unknown. By alignment of telomerase RNAs of Saccharomyces cerevisiae and six Kluyveromyces species followed by mutagenesis of the S. cerevisiae RNA, we found a conserved region that is essential for telomere maintenance. Phylogenetic analysis and computer folding revealed that this region is conserved not only in primary nucleotide sequence but also in secondary structure. A common bulged-stem structure was predicted in all seven yeast species. Mutational analysis showed the structure to be essential for telomerase function. Suppression of bulged-stem mutant phenotypes by overexpression of Est1p and loss of co-immunoprecipitation of the mutant RNAs with Est1p indicated that this bulged stem is necessary for association of Est1p, a telomerase regulatory subunit. Est1p in yeast extract bound specifically to a small RNA containing the bulged stem, suggesting a direct interaction. We propose that this RNA structure links the enzymatic core of telomerase with Est1p, thereby allowing Est1p to recruit or activate telomerase at the telomere.

Conserved Sequence↗