Investigating RNA folding and chaperone activity by multidimensional NMR spectroscopy

The aim of this project is to provide a better understanding of the folding free-energy landscape of ribonucleic acids (RNA), and the role of a specific RNA fold for biological function. The folding landscape of RNAs is rugged and beside the global minimum energetically close suboptimal folding states are often found and populated. As a consequence of this rugged energy landscape, RNAs can easily become trapped in non-functional conformations. Our study will focus on two particular questions: (1) what are the molecular bases for the conformational transitions that riboswitch RNAs undergo as a response to changing environmental conditions in order to induce or abolish expression of a particular gene? (2) What are the molecular mechanisms governing the activity of RNA chaperon proteins that assist in the folding process of RNA? We will use and further develop fast high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy methods to study the processes of RNA folding, ligand binding, and ligand-induced conformational transitions in real time. This will yield atomic-resolution information on the kinetics of the structural transitions, and the transient population of eventual intermediate states. In addition, we will investigate the presence of low populated excited state conformations under equilibrium conditions using spin relaxation and hydrogen exchange NMR experiments. The experimental NMR work will be aided by the development of site-specific 13C isotope labeling schemes that provide the high spectral resolution required for NMR studies of larger RNA molecules, and increase the number of sites available for probing local structure and dynamics. We have selected a number of target RNA molecules, and RNA chaperone proteins that will be produced, isotope-labeled and investigated by NMR spectroscopy during this project.

We successfully implemented a way to address dynamic phenomena of RNA using a combination of chemical, biological and physical methods that allows us to visualize RNA function with unprecedented precision. RNA is no longer regarded as the small brother of DNA. Its recently discovered functions in regulatory networks of cells led to significant scientific efforts to understand structure and dynamics of RNA on a molecular level. Using a highly interdisciplinary approach by combining organic synthesis and biochemicial and biophysical methods we are now able to record molecular movies of RNA in action.