52 Examining the effects of 2′-OH substitutions on the structure and stability of the S. cerevisiae telomerase RNA pseudoknot and tertiary structure

Abstract

Structural and functional characterization of the pseudoknot in the Saccharomyces cerevisiae telomerase RNA (TLC1) demonstrated that tertiary structural interactions occur between loop 1 uridines and stem 2 Watson–Crick A-U pairs, as previously observed for the K. lactis and human telomerase RNA pseudoknots. The contributions of backbone groups in the pseudoknot to telomerase catalysis were investigated using 2′-OH (ribose) to 2′-H (deoxyribose) substitutions and 2′-O methylation at specific nucleotides within the stem 2 pseudoknot region (Huang & Yu, 2010; Qiao & Cech, 2008). Based on investigations of the structural and thermodynamic properties of the TLC1 RNA pseudoknot region, which provided a more detailed description of the secondary structure of the pseudoknot stem 2 helical region (Liu et al., 2012), including an additional upstream stem 2 base-paired sequence, we examined the structural and thermodynamic perturbations of the 2′-O methyl and 2′-H substituted pseudoknots using UV-monitored thermal denaturation experiments, native gel electrophoresis, CD spectroscopy, and nuclear magnetic resonance spectroscopy (Liu & Theimer, 2012). These results show a correlation between A-form RNA geometry, thermodynamic stability, and telomerase activity in the triple helix substitutions, and are consistent with the identification of the U809 2′-OH as a contributor to telomerase activity. We have since extended these observations to more completely characterize the effects of additional substitution types and positions in the pseudoknot and tertiary structure to obtain greater insight into thermodynamic, structural, and functional consequences of 2′-OH substitutions in this important secondary and tertiary structural element.