Single-Molecule Analysis of the Functional Structure of Telomerase RNP

Abstract

Telomerase is a cellular ribonucleoprotein (RNP) complex comprised of telomerase RNA, telomerase reverse transcriptase (TERT) and other protein cofactors. The activity of the telomerase enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report the development and application of a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from individual enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. In particular, we have established that the phylogenetically conserved pseudoknot motif within telomerase RNA is properly folded in catalytically active enzymes. Interestingly, the pseudoknot motif is misfolded in the absence of proteins, and the protein subunits of the telomerase holoenzyme counteract RNA 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 various telomerase RNA regions such as the pseudoknot but also establish a direct method for elucidating how the structure of telomerase enables its function.