Telomerase limits the extent of base pairing between template RNA and telomeric DNA

Abstract

Telomerase is the ribonucleoprotein reverse transcriptase that adds telomeric DNA repeats to the ends of chromosomes. This involves annealing of the telomerase RNA template to the 3′ end of the chromosome, reverse transcription of the RNA template by the telomerase reverse transcriptase polypeptide and translocation. Here, we overexpress and partially purify the catalytically active yeast telomerase core in its natural host and probe telomerase RNA base methylation accessibility with dimethyl sulphate in the presence and absence of a DNA substrate and after substrate elongation. The length of the RNA-DNA hybrid is kept constant at seven base pairs after primer binding and elongation. Thus, new base-pair formation at the 3′ end of the substrate during elongation coincides with disruption of base-pair interactions at the other side of the template. Presumably, this circumvents the generation of an exceedingly high energy barrier for translocation and dissociation. Our analysis also corroborates recently proposed yeast telomerase RNA secondary structure models. ©2005 European Molecular Biology Organization.