Book of Abstracts: Albany 2005
Site-Resolved Dynamics and Energetics of a Ribosomal RNA
The stability and dynamics of individual base pairs in a 29-nt RNA molecule has been investigated by proton exchange and nuclear magnetic resonance (NMR) spectroscopy. The RNA nucleotide sequence (5'-G1 G2 G3 U4 G5 C6 U7 C8 A9 G10 U11 A12 C13 G14 A15 G16 A17 G18 G19 A20 A21 C22 C23 G24 C25 A26 C27 C28 C29-3') is that of the rat 28S rRNA sarcin-ricin loop (SRL), and includes the 12-nt sequence (5'-AGUACGAGAGGA-3') that is conserved in all ribosomal RNAs. The loop is named for α-sarcin and ricin, two protein toxins that kill eukayotic cells by catalyzing the cleavage of bonds within the RNA: α-sarcin cleaves the phosphodiester bond between G16 and A17, and ricin and its analogs depurinate A15 of the consensus sequence. This process is lethal for the cells because neither enlongation factor 1α nor factor 2 can interact normally with a ribosome when SRL is modified by either toxin. The structure of the 29-nt SRL RNA has been previously determined by NMR (1) and by crystallography at 2.1 Å resolution (2). The molecule consists of the following structural motifs: a typical A-form stem part (G1-C6 and G24-C29); a flexible connector region (U7-A9 and A21-C23); a bulged G-motif including a GUA base triple (G10, U11 and A20) and a side-by-side G-A base pair (G19 and A12); a cross-strand G-stacking motif (G10, G19, G18 and G14); and a GNRA tetraloop (G14, A15, G16, A17). Therefore, this molecule provides a platform for investigating several RNA structural motifs within a single RNA molecule. In the present work we have mapped the stability of the structural motifs in SRL RNA by NMR. The exchange rates of imino protons were measured as a function of the concentration of exchange catalyst (ammonia). These exchange rates were used to obtain the rates and the equilibrium constants for opening of individual base pairs. The opening equilibrium constants provided the free energies of stabilization of various parts of the structure. The relationship between these results and the structure of each RNA motif will be discussed.
References and Footnotes
1Department of Chemistry and Molecular Biophysics Program