Abstract
Telomerase is a unique reverse transcriptase that maintains the 3′ ends of eukaryotic chromosomes by adding tandem telomeric repeats. The RNA subunit (TR) of vertebrate telomerase provides a template for reverse transcription, contained within the conserved template/pseudoknot domain, and a conserved regions 4 and 5 (CR4/5) domain, all essential for catalytic activity. We report the nuclear magnetic resonance (NMR) solution structure of the full-length CR4/5 domain from the teleost fish medaka (Oryzias latipes). Three helices emanate from a structured internal loop, forming a Y-shaped structure, where helix P6 stacks on P5 and helix P6.1 points away from P6. The relative orientations of the three helices are Mg2+ dependent and dynamic. Although the three-way junction is structured and has unexpected base pairs, telomerase activity assays with nucleotide substitutions and deletions in CR4/5 indicate that none of these are essential for activity. The results suggest that the junction is likely to change conformation in complex with telomerase reverse transcriptase and that it provides a flexible scaffold that allows P6 and P6.1 to correctly fold and interact with telomerase reverse transcriptase.
Molecular dynamics correctly models the unusual major conformation of the GAGU RNA internal loop and with NMR reveals an unusual minor conformation