Book of Abstracts: Albany 2007
June 19-23 2007
Cooperative and pH Effects of Pseudouridine on Structure and Stability in Helix 69 of Escherichia coli 23 S rRNA
Helix 69 of 23 S rRNA is a region of considerable interest. It is located at the interface of the two ribosomal subunits. It has been suggested that H69 plays an essential role in subunit association and ribosomal release factor dependent translation termination. Furthermore, helix 69 is believed to be a dynamic region of the ribosome, participating in RNA-RNA and RNA-protein interactions. Helix 69 in Escherichia coli contains three modifications, two pseudouridines (Ψ) and a 3-methylpseudouridine. Pseudouridine, the most abundant modification in rRNA, can exist in different nucleoside conformations. Energy barriers between possible conformations of pseudouridine are low, such that conformational changes are possible with small energy perturbations. Experiments have been carried out to investigate the dynamic nature of helix 69. Circular dichroism spectroscopic studies and thermal melting experiments of helix 69 RNA constructs have revealed pH-dependent structural and stability changes. Constructs having pseudouridines replaced with uridines do not show structural changes, emphasizing the importance of pseudouridine in helix 69. NMR spectroscopy of helix 69 suggests that a protonation event may occur at or near the Ψ1911 at the loop closing base pair position. The remaining two pseudouridines, Ψ1915 and Ψ1917 which are located in the loop region, also cause show structural perturbations. Spectroscopic and thermodynamic experiments have enabled us to determine cooperative structural and stability effects between pseudouridines of helix 69, which may assist in propagation of structural perturbations to the loop region in helix 69.