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.
Seto
Anita G
AG
Department of Chemistry and Biochemistry, and Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA.
Umansky
Kfir
K
Tzfati
Yehuda
Y
Zaug
Arthur J
AJ
Blackburn
Elizabeth H
EH
Cech
Thomas R
TR
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D009154:Mutation; D012331:RNA, Fungal / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic
D017931:DNA Primers; D012331:RNA, Fungal; D019098:Telomerase