Structural dynamics of the human telomerase RNA pseudoknot

The essential telomerase enzyme adds telomere DNA repeats to chromosome ends, combating chromosome degradation and erroneous DNA repair processes. The telomerase catalytic core consists of the telomerase reverse transcriptase protein (hTERT) and telomerase RNA (hTR). hTR provides the template sequence for the reverse transcription reaction and serves as a scaffold for other telomerase-associated proteins. Among several conserved structural motifs within hTR, the structure of a pseudoknot fold that undergoes compaction into a triple helical structure has been solved using NMR. Pathogenic mutations within the hTR pseudoknot domain perturb the structural stability of this complex RNA fold. Previous structural studies of the hTR pseudoknot employed minimal RNA constructs to facilitate experimental analysis. Therefore, we established a single-molecule FRET based assay to characterize the structural dynamics of the hTR pseudoknot in the context of the native RNA sequence. Our results suggest the folding properties of the hTR pseudoknot are significantly influenced by the presence of proximal RNA structural elements. As anticipated we find the stability of the hTR pseudoknot to be highly sensitive to Mg(II) concentration. Lastly, we demonstrate that the structural manifestation of a specific pathogenic mutation within the hTR pseudoknot depends on both the sequence context and Mg(II) concentration. Our results demonstrate that RNA pseudoknot formation in the functional hTR sequence deviates from its behavior in minimal constructs. Our approach permits real-time analysis of the functional hTR structure and may thus pave the way for future studies on structural dynamics of human telomerase.