A stem-loop of Tetrahymena telomerase RNA distant from the template potentiates RNA folding and telomerase activity

The ribonucleoprotein enzyme telomerase adds telomeric repeats to the ends of linear chromosomes. The Tetrahymena telomerase reverse transcriptase (TE RT) protein and the telomerase RNA can be reconstituted into an active comp iler in vitro in rabbit reticulocyte lysates. We have probed the structure of the telomerase RNA in the reconstituted complex with RNases T1 and V1. U pon TERT binding to the RNA, sites of both protection and enhancement of cl eavage were observed, suggesting potential protein-binding sites and confor mational changes in the RNA. Especially prominent was a large region of RNa se VI protection in stem-loop IV. A number of loop IV mutants still bound T ERT but showed drastic decreases in the level of telomerase activity and th e loss of protein-dependent folding of the pseudoknot region of the telomer ase RNA. The telomerase activity defect and the misfolding of the pseudokno t were partially separable, leading to the proposal of two functions for st em-loop IV: to aid in the folding of the pseudoknot and to function more di rectly in the active site of telomerase. Thus an RNA element far from the t emplate makes a major contribution to Tetrahymena telomerase enzyme activit y.