Structural basis for ligand specificity and affinity in naturally occuring and artificial purine-binding RNA molecules
Zusammenfassung der Projektergebnisse
Not all aims of the original proposal could be reached in the course of our work. In particular, we were unable with our solution NMR approach to compete successfully against X-ray crystallography for the structure determination of the class I-A 2’-dG riboswitch bound to its ligand. On the other hand, using solution NMR we were able to resolve important structural features of the ligand free-state of this riboswitch. However, we were able to delineate the degree of preformation of tertiary structure with respect to long-range loop-loop interactions and compare them with the structural preformation observed in the wild-type and mutant purine riboswitches. Furthermore, using time-resolved NMR-spectroscopy we were able to show that purine and class I-A 2’-dG riboswitches utilize different approaches to distinguish cognate and non-cognate ligands based either one the kinetics or the thermodynamics of ligand binding. On the other hand, our NMR-studies of artificial aptamer-ligand complexes revealed a much richer structural landscape of GTP-binding motifs than previously anticipated. Here, NMR-spectroscopy was crucial in delineating unexpected structural features of the ligand-binding site such as the presence of protonated adenine residues in tertiary structure elements due to the direct observability of protons in our NMR-experiments. Furthermore, we demonstrated that NMR-spectroscopy is able to derive interesting insights into ligand binding modes even in the absence of a full structure determination as in the case of the class-V aptamer. Our structural results obtained for the GTP-aptamers not only contribute to a better appreciation of the structural variability of ligand binding motifs in complex RNA but will hopefully also contribute to a reassessment of the importance of stably protonated nucleotides for the architecture of complex RNA structural elements. The possibility that protonated nucleotides contribute to RNA tertiary structures at ambient pH is so far largely overlooked in particular in the context of RNA structural modelling efforts.
Projektbezogene Publikationen (Auswahl)
- Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain. Nucleic Acids Res. 2010, 38: 4143-4153
J. Buck, J. Noeske, J. Wöhnert, H. Schwalbe
- Influence of ground-state structure and Mg2+ binding on folding kinetics of the guaninesensing riboswitch aptamer domain. Nucleic Acids Res. 2011, 39: 9768-9778
J. Buck, A. Wacker, E. Warkenthin, J. Wöhnert, J. Wirmer-Bartoschek, H. Schwalbe
- Structure and dynamics of the deoxyguanosine-sensing riboswitch studied by NMR-spectroscopy. Nucleic Acids Res. 2011, 39: 6902-6812
A. Wacker, J. Buck, D. Mathieu, C. Richter, J. Wöhnert, H. Schwalbe
- Mechanisms for differentiation between cognate and near-cognate ligands by purine riboswitches. RNA Biology 2012, 8: 672-680
A. Wacker, J. Buck, C. Richter, H. Schwalbe, J. Wöhnert
(Siehe online unter https://doi.org/10.4161/rna.20106)
