MUTATIONAL ANALYSIS OF THE TETRAHYMENA TELOMERASE RNA - IDENTIFICATION OF RESIDUES AFFECTING TELOMERASE ACTIVITY IN-VITRO

Telomere-specific repeat sequences are essential for chromosome end st ability. Telomerase maintains telomere length by adding sequences de n ovo onto chromosome ends. The template domain of the telomerase RNA co mponent dictates synthesis of species-specific telomeric repeats and o ther regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides i n the RNA component that are important for telomerase activity. Althou gh many changes to conserved features in the RNA secondary structure d id not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role i n telomerase function. The template boundary was only altered by chang es in stem II that affected the conserved region upstream of the templ ate, not by changes in other regions, such as stems I, III and IV, con sistent with a role of the conserved region in defining the 5' boundar y of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests t hat this base pairing interaction may not be required for telomerase a ctivity per se but may be conserved as a regulatory site for the enzym e in vivo.