Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA

Abstract

Telomerase is a unique ribonucleoprotein complex that catalyzes the addition of telomeric DNA repeats onto the 3' ends of linear chromosomes. All vertebrate telomerase RNAs contain a catalytically essential core domain that includes the template and a pseudoknot with extended helical subdomains. Within these helical regions is an asymmetric 5-nt internal bulge loop (J2a/b) flanked by helices (P2a and P2b) that is highly conserved in its location but not sequence. NMR structure determination reveals that J2a/b forms a defined S-shape and creates an ∼90 ° bend with a surprisingly low twist (∼10 °) between the flanking helices. A search of RNA structures revealed only one other example of a 5-nt bulge, from hepatitis C virus internal ribosome entry site, with a different sequence but the same structure. J2a/b is intrinsically flexible but the interhelical motions across the loop are remarkably restricted. Nucleotide substitutions in J2a/b that affect the bend angle, direction, and interhelical dynamics are correlated with telomerase activity. Based on the structures of P2ab (J2a/b and flanking helices), the conserved region of the pseudoknot (P2b/P3, previously determined) and the remaining helical segment (P2a.1-J2a.1 refined using residual dipolar couplings and the modeling program MC-Sym) we have calculated an NMR-based model of the full-length pseudoknot. The model and dynamics analysis show that J2a/b serves as a dominant structural and dynamical element in defining the overall topology of the core domain, and suggest that interhelical motions in P2ab facilitate nucleotide addition along the template and template translocation.