Figure 4.

Effect of primer-template hybrid formation on FRET distributions. Primers capable of forming 5–9 basepairs with template RNA were tested in smFRET telomerase binding assays with U36-labeled telomerase. (A) Schematic diagram of the docked state for all six primers used in smFRET experiments, demonstrating the number of basepairs formed and the expansion of the template RNA as the RNA–DNA duplex becomes progressively longer (22). (B) smFRET histograms for wild-type enzyme. As primers contain progressively more telomeric DNA sequence, the predominant FRET distribution of the docked state shifts from ∼0.75 FRET to ∼0.5 FRET. In addition, the ∼0.25 FRET alternative state disappears. (C) smFRET histograms for L14A mutant enzyme.