Studying functional RNA dynamics by NMR spectroscopy

Conformational transitions in RNA stem-loop structures are the basis of functionally important transitions in large RNA molecules. The focus of this project is to create experimental access to the characterization of the dynamics of these fundamental RNA folding modules using nuclear magnetic resonance spectroscopy (NMR). NMR techniques provide atom-resolved information and thus offer the possibility to observe dynamic processes at various positions, which is of fundamental importance, since dynamic properties are not necessarily equally distributed within a biological macromolecule. I am planning to apply NMR spin relaxation techniques that have been developed for proteins to the field of RNA chemistry and biophysics, design new NMR pulse sequences and combine spectroscopic methods with selective labelling techniques. This approach will enable me to gain detailed insight into the reactivity and dynamics of various RNA model systems. These model systems will cover a broad range of questions: for instance, NMR active isotopes can be introduced at strategic positions in bistable RNA molecules in order to characterize the spontaneous transitions between the stable structures. Another part of the project is concerned with elucidating at atomic resolution the mechanisms of reactions between RNA molecules by altering the primary sequences of the reaction partners. Another question concerns the effect of the bacterial RNA binding protein CspA, which is essential for maintaining correctly folded RNA under coldshock conditions, on the kinetics of the proposed reactions. The results will provide important insight into the mechanism of action of this important protein. According to the question of interest, motions on different time-scales will be addressed: bond vector librations (picosecond time range), molecular diffusion (nanoseconds), conformational exchange (milliseconds to microseconds), spontaneous transitions between different stable structures (on the order of seconds), and chemical reactions such as duplex formation or strand exchange (tens of seconds to minutes). Within this project I will provide the theoretical framework that is necessary to design new experiments and correctly interpret the obtained data including computation of relaxation rates in methyl groups, description of the kinetics of complex reaction mechanisms, as well as analysis of relaxation measurements in multi-state processes). With these studies I attempt to contribute an important step towards a comprehensive picture of functional RNA dynamics in solution.

The central dogma of molecular biology has, for a long time, seen the main function of RNA as a translator passing down sequential genetic information between DNA and the ultimate executors of cellular functions, the proteins. Recently this view of RNA function has been challenged by the observation of so-called small or non-coding RNAs that are crucial for a plethora of regulatory tasks on the level of replication, transcription, and translation, revealing how the flow of information between the canonical up- and downstream molecular species is embedded in a network which connects it to environmental signals.On a molecular basis, many of these functions are linked to the special structural and dynamic properties of RNAs for instance, RNA molecules signalling the necessity to switch on (or off) a particular transcriptional pathway in response to metabolic conditions (so-called riboswitches) do so by accessing different conformations a feature that makes them natural targets of NMR spectroscopy.Such structural versatility is reflected in the particular energy surface, which is similar to a map where a stable structure appears as a minimum. For small RNA molecules several nearly isoenergetic minima (corresponding to the different conformations) may exist, with a barrier beween them that determines the frequency of transitions. For RNAs investigated in this project, rates of interconversion were on the order of one to several thousand per second an instantaneous reaction on the time-scale of a mileu change in a cell (guaranteeing proper function), and at the same time representing the very window for dynamics observation by NMR spectroscopy. In such cases it is possible to reconstruct the energetic landscape of functionally relevant degrees of freedom of a molecular scaffold.This project took on the task to study such interconversions on model systems as well as biologically relevant RNAs using existing and novel NMR methodology in conjunction with isotope labelling schemes developed at the Institute of Organic Chemistry in Innsbruck. Using a variety of NMR methods, it was demonstrated that it is possible to obtain structural information about as well as rates of interconversion between RNA conformations (even in the presence of composite dynamic processes). From temperature dependent data it was possible to quantitatively determine the thermodynamics and activation parameters of several investigated processes, and, with this, to obtain information on the mechanisms of interconversion.