Tetrahymena telomerase ribonucleoprotein RNA±protein interactions

Telomerase is an enzyme that is essential for the replication and maintenance of chromosomal termini. It is a ribonucleoprotein consisting of a catalytic subunit, one or more associated proteins, and an integral RNA subunit that serves as a template for the synthesis of telomeric repeats. We identified a Tetrahymena telomerase RNA–protein complex by an electrophoretic mobility shift assay, using telomerase partially purified from whole cell extracts and radiolabeled, in vitro transcribed wild-type Tetrahymena telomerase RNA. Complex formation was specific as unlabeled Tetra-hymena telomerase RNA, but not Escherichia coli ribo-somal RNAs, competitively inhibited complex formation. Binding required concentrations of MgCl2 of at least 10 mM and occurred over a wide range of potassium glutamate concentrations (20–220 mM). The RNA– protein complex was optimally reconstituted with a 30C preincubation for ≤5 min, prior to electrophoresis. Certain Tetrahymena telomerase RNAs containing deletions of structures and sequences previously pre-dicted to be involved in protein binding were unable to competitively and specifically inhibit complex formation, suggesting a role in protein binding for the deleted residues or structures