Structural analysis of the catalytic core of human telomerase RNA by FRET and molecular modeling

Abstract

Telomerase is the ribonucleoprotein reverse transcriptase involved in the maintenance of the telomeres, the termini of eukaryotic chromosomes. The RNA component of human telomerase (hTR) consists of 451 nucleotides with the 5' half folding into a highly conserved catalytic core comprising the template region and an adjacent pseudoknot domain (nucleotides 1-208). While the secondary structure of hTR is established, there is little understanding of its three-dimensional (3D) architecture. Here, we have used fluorescence resonance energy transfer (FRET) between fluorescently labelled peptide nucleic acids, hybridized to defined single stranded regions of full length hTR, to evaluate long-range distances. Using molecular modeling, the distance constraints derived by FRET were subsequently used, together with the known secondary structure, to generate a 3D model of the catalytic core of hTR. An overlay of a large set of models generated has provided a low-resolution structure (6.5-8.0 A) that can readily be refined as new structural information becomes available. A notable feature of the modeled structure is the positioning of the template adjacent to the pseudoknot, which brings a number of conserved nucleotides close in space.

Figure 1

Secondary structure of human telomerase RNA (hTR) (adapted from refs and ( and 4)). The underlined sequence around the 5′ end, the template, and in loop J2a/3 of hTR indicate the PNA binding sites (for PNA1, PNA2, and PNA3, respectively). FRET distances determined in this study are indicated by dotted lines.

Figure 2

Fluorescence data for a typical FRET experiment using the PNA2nTMR/PNA3cFl molecular pair. The emission spectra are as follows: fluorescein- and TMR-labeled hTR (donor-acceptor, solid line) and fluorescein-labeled hTR (donor only, broken line). The blank measurement (unlabeled hTR, dashed line) was used for background correction. Transfer efficiency values (E) were calculated using eq 2.

Figure 3

Superimposition of RNA helices in lowest energy refined models. (a) Superimposition of helices of the catalytic core of human telomerase RNA (cluster I, 21 models). For clarity, the helical elements are displayed as cylinders of appropriate length but with diameter 50% that of an A-form RNA helix width. (b) Variation in the position of helices between the superimposed structures of cluster I. The variation is expressed as the mean RMSD (​<​RMSD​>​) in the position of a particular helix midpoint relative to the corresponding helix in the lowest energy structure. The average of the RMSD values for all helices (6.5 Å) is represented by the dotted line. P2b was actually fitted to two helical segments whose displacements were averaged for presentation in this graph.

Figure 4

Schematic representation of lowest energy model. (a) Refined 3D molecular model of nucleotides 1–208 of human telomerase RNA. The RNA backbones are represented as ribbons colored gray for linkers and PNA or distinctive colors for each helix. Base positions in helices are indicated schematically by single colored sticks positioned by the nuccyl program. Nucleotides invariant among all vertebrates are shown as red spheres. The green sphere corresponds to the position of the 3′ end of the RNA representing PNA2 and, thus, the 3′ end of the DNA primer immediately after a repeat addition but prior to translocation. (b) Alternative view of structure after a 90° rotation about the vertical axis.

Figure 5

Comparison of refined pseudoknot with NMR model. Superimposition of the backbone structure of hTR pseudoknot, derived by NMR spectroscopy (yellow), with that of our further refined model (orange). The loop spanning nt 122–170, shown in gray, is a particular feature of the RNA used in the NMR studies (12) and was not included in the RMSD calculation.