A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro.
The ribonucleoprotein enzyme telomerase synthesizes DNA at the ends of chromosomes. Although the telomerase catalytic protein subunit (TERT) is well conserved, the RNA component is rapidly evolving in both size and sequence. Here, we reduce the 1,157-nucleotide (nt) Saccharomyces cerevisiae TLC1 RNA to a size smaller than the 451-nt human RNA while retaining function in vivo. We conclude that long protein-binding arms are not essential for the RNA to serve its scaffolding function. Although viable, cells expressing Mini-T have shortened telomeres and reduced fitness as compared to wild-type cells, suggesting why the larger RNA has evolved. Previous attempts to reconstitute telomerase activity in vitro using TLC1 and yeast TERT (Est2p) have been unsuccessful. We find that substitution of Mini-T for wild-type TLC1 in a reconstituted system yields robust activity, allowing the contributions of individual yeast telomerase components to be directly assessed.
Zappulla
David C
DC
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Goodrich
Karen
K
Cech
Thomas R
TR
D001483:Base Sequence; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase