NMR analysis of the thymidylate synthase mRNA binding site 1-paromomycin interaction, Moorella thermoacetica selenocysteine elongation factor binding surface for its SECIS RNA, and the nucleocapsid hairpin loop structure of the Ebola virus

1. Structural elucidation of the thymidylate synthase mRNA binding site 1 construct (TSMC) and investigation of the paromomycin-TSMC interaction via NMR analysis. The chemical shift perturbations of the H6/H5 protons of TSMC upon paromomycin binding were analyzed via TOCSY experiments. The sum of incremental displacements in delta1H H5 and delta1HH6 for cytosine residues monitoring the high and low affinity binding of paromomycin were fit to a sequential two site binding model. The specific and non-specific dissociation constants were calculated 101 +/- 79.1 muM, and 706 +/- 301 muM, respectively. The CC mismatch and its surrounding GC basepairs experienced the highest degree of chemical shift change. Specifically, paromomycin approached the major groove side of these residues. The solution state structure of TSMC free was determined. The CC mismatch is stabilized by a non-canonical one hydrogen bond basepair and its hydrogen bonding pattern reveals its structural relationship with its flanking GC basepairs. Regarding paromomycin, rings I and II exhibit the highest degree of chemical shift change.;3. NMR evidence of an internal basepair in the 'pseudoheptaloop' of the Ebola viral Nucleocapsid Hairpin. The most studied protein of the Ebola genome, VP30, has a transcriptional activation role on the first gene of this virus. This biochemical event is important for regulating levels of a nucleoprotein. Specifically, VP30 is thought to interact with an AU rich 'seven-membered' loop of this nucleocapsid hairpin. Complete proton assignments of this RNA, including those obtained from hydrogen bond experiments were used to identify an internal AU basepair in this 'pseudoheptaloop'.;2. A study of the Moorella thermoacetica SelB/SECIS hairpin interaction via NMR analysis. Measurement of the chemical shift changes of SelB-MT508--634 (i.e. Winged Helix Domain (WHD) 3 and 4) due to SECIS-MT RNA binding was sufficient to map out the binding surface of this protein. NMR data also showed a region in the 3rd WHD that was affected by SECIS-MT binding. This secondary site is located at exactly a position where a bulged out uridine residue of the E. coli SECIS RNA would make a closer contact. This was used to propose the higher affinity observed for the SelB-EC/SECIS-EC complex over the SelB-MT/SECIS-MT pair.