The structure of an enzyme-activating fragment of human telomerase RNA

Abstract

The ribonucleoprotein enzyme telomerase ensures the stability and fidelity of linear chromosome ends by elongating the telomeric DNA that is lost during each round of DNA replication. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence, acts as the scaffold for ribonucleoprotein assembly, and activates the enzyme for catalysis. Vertebrate telomerase RNAs contain three highly conserved structural and functional domains: the template domain, the "CR4-CR5" or "activation" domain essential for activation of the enzymatic activity, and a 3'-terminal "box H/ACA"-homology domain responsible for ribonucleprotein assembly and maturation. Here we report the NMR structure of a functionally essential RNA structural element derived from the human telomerase RNA CR4-CR5 domain. This RNA, referred to as hTR J6, forms a stable hairpin interrupted by a single nucleotide bulge and an asymmetric internal loop. Previous work on telomerase has shown that deletion of the hTR J6 asymmetric internal loop results in an RNA incapable of binding the enzymatic protein component of the RNP and therefore an inactive RNP without telomerase activity. We demonstrate here that the J6 internal loop introduces a twist in the RNA structure that may position the entire domain into the catalytic site of the enzyme.

FIGURE 1.

(A) A schematic of the secondary structure and domain structure of human telomerase RNA. The template, H/ACA, and CR4-CR5 domains are indicated by blue, green, and red boxes, respectively. (B) The sequence of the RNA studied and regions of secondary structure are indicated as paired regions 6a and 6b (P6a and P6b) and bulge loop 6 (L6). The numbering scheme matches that of wild-type hTR.

FIGURE 2.

NMR spectra of the hTR J6 containing RNA. (A) The anomeric to aromatic region of a 300-msec mixing time NOESY recorded at 750 MHz with sequential H1′ to aromatic peaks labeled “s,” intraresidue peaks are labeled “i,” and H5/6 cross-peaks for the pyrimidines are indicated in gray. (B) The aromatic region of a constant time ¹³C HSQC.

FIGURE 3.

Superimposition of the 10 best-scoring members of the ensemble of hTR J6 RNA structures. Residues are colored according to the diagram in Figure 1B, except that the backbone is traced in grey to highlight the distorted curvature and ζ-like conformation induced by the J6 internal loop.

FIGURE 4.

(A) Stereo views of the J6 bulge region from the lowest energy conformer of the hTR J6 structure, highlighting the juxtaposition of C266 and U291, the putative base triple (indicated by a red arrow), and the stacking of A289 and C290 under the P6b stem. (B) A superimposition of the nucleotides involved in this proposed base triple clearly shows the two populations of the C267 position (red arrow) for the 10-structure ensemble, with the less frequently observed stacked conformation displayed in pale green. Note that the perspective for B is rotated by ∼180° with respect to A. (C) The C266/U291 pair consistent with phylogenetic conservation is shown with possible hydrogen bonds indicated by the red dashed lines. (D) A possible hydrogen-bonding network stabilizing the base triple is indicated by dashed red lines.

FIGURE 5.

Two schematic views of the structure derived from the output of the CURVES program (Lavery and Sklenar 1988), rendered with RASMOL (Sayle and Milnerwhite 1995), rotated by ∼90° with respect to each other. These images highlight the angular bend (A; 20°) and deflection (B; 3 Å) between the two stem segments, P6a and P6b, induced by the internal loop J6.

FIGURE 6.

The solvent accessible surface of the hTR J6 structure, showing a tunnel through the core of the J6 region. The N3 atom of C290 (red) is only reactive to dimethyl sulfate (DMS) in the absence of the hTERT protein (Antal et al. 2002). This region is a likely binding determinant for hTERT, and we speculate that this cavity is a protein-docking site.

FIGURE 7.

Progress toward the complete NMR structure of the full CR4/CR5 domain. Atomic coordinates replace nucleotide sequence for the two regions we have determined by NMR, and represent 64% of the functional sequence (47/73 nucleotides). Residues with ≥80% sequence conservation are colored red; many of these residues are present in the RNA structures that we have determined.