Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.