Structural energetics and dynamics of nucleic acids and their interactions with metal ions

This thesis presents investigations of structural energetics and dynamics of nucleic acids and their interactions with metal ions by Nuclear Magnetic Resonance (NMR) spectroscopy.;The first part of the thesis describes a characterization of the energetics and dynamics of TATA box DNA at the level of individual base pairs. The TATA box DNA dodecamer investigated in this thesis (5'-GCTATAAAAGGG-3'/5'-CCCTTTTATAGC-3') contains the same TATA element as that in the Adenovirus major late promoter. The results indicate that TATA box DNA has an intrinsic energetic asymmetry, which may contribute to the binding directionality of TATA box-binding protein. The opening of AT/TA base pairs in various base-step configurations is also being examined in this AT-rich DNA dodecamer. The results indicate that the opening reactions of AT/TA base pairs in TATA box DNA have a strong dependence on the location and the sequence context of each base pair.;The second part of the thesis describes two investigations of Sarcin-Ricin Domain RNA: (1) characterization of the structural energetics and opening dynamics of individual bases and base pairs; (2) mapping divalent metal ion binding sites. Imino proton exchange measurements reveal that the structural stabilization free energies are broadly distributed in this RNA structure. One of the least stable sites is the noncanonical G-A base pair located next to the phosphodiester bond cleaved by alpha-sarcin. This finding supports the proposed base flipping mechanism for alpha-sarcin cleavage. Our results also indicate that the bases in the consensus loop have relatively long lifetimes in the open state. Mn2+ titration experiments demonstrate that of two specific Mn2+ ion binding sites exist in Sarcin-Ricin Domain RNA. One is located in the major groove of the stem part and the other one is located in the tetraloop.;The third part of the thesis presents a characterization of structural energetics of a DNA/RNA hybrid from an intrinsic transcription terminator site. Our preliminary results show that the rG-dC, dG-rC and rA-dT base pairs of the hybrid have similar stability to those in A-form RNA duplexes, whereas the dA-rU base pairs are very unstable compared with rA-rU base pairs in RNA duplexes. The dA-rU base pairs also destabilize substantially the neighboring base pairs.