The solution structure of an essential stem-loop of human telomerase RNA

Abstract

The ribonucleoprotein enzyme telomerase maintains chromosome ends in most eukaryotes and is critical for a cell's genetic stability and its proliferative viability. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence as well as a scaffold for ribonucleoprotein assembly. Vertebrate telomerase RNAs have three essential domains: the template, activation and stability domains. Here we report the NMR structure of an essential RNA element derived from the human telomerase RNA activation domain. The sequence forms a stem-loop structure stabilized by a GU wobble pair formed by two of the five unpaired residues capping a short double helical region. The remaining three loop residues are in a well-defined conformation and form phosphate-base stacking interactions reminiscent of other RNA loop structures. Mutations of these unpaired nucleotides abolish enzymatic activity. The structure rationalizes a number of biochemical observations, and allows us to propose how the loop may function in the telomerase catalytic cycle. The pre-formed structure of the loop exposes the bases of these three essential nucleotides and positions them to interact with other RNA sequences within TR, with the reverse transcriptase or with the newly synthesized telomeric DNA strand. The functional role of this stem-loop appears to be conserved in even distantly related organisms such as yeast and ciliates.

Figure 1

(a) Secondary structure of human telomerase RNA with the template (blue), activation (red) and stability (green) domains. The minimal activation domain sequence is shown. (b) Proposed secondary structure and sequence of the P6.1 RNA construct studied here. Residues are numbered from 301 to 315 throughout the text and figures to match the numbering in hTR.

Figure 2

(a) 800 MHz NOESY spectrum shows high dispersion indicative of a well-defined structure. (b) 500 MHz ¹³C ct-HSQC spectrum; ¹H-¹³C correlations are labeled for 1′ to 4′ spin systems.

Figure 3

(a) Superposition of 36 converged structures based on the stem residues. (b) Superposition of residues U306 to G310; the 5′ phosphorus of G309 is indicated by a sphere. (c) A representative structure of the 5 nt P6.1 loop and closing CG pair is shown to highlight the exposure of the three central loop bases U307 to G309.

Figure 4

T₁ρ relaxation parameters measured for base ¹H/¹³C resonances of P6.1 as described previously (42). It is notable that the values observed for loop residues are only slightly higher than for stem residues, indicating that the loop structure is nearly as restricted motionally as the double helical stem.

Figure 5

(a) Structure of the five loop residues from the P6.1 structure (heavy atoms only); (b) the four loop residues of the UUCG tetraloop (49). Color-coding highlights residues in analogous positioning regardless of residue type. The red residues are the 5′ uracyls from both sequences and the grey residues are at the 3′ ends of the loops.