Dynamics of RNA under the influence of RNA chaperones

Context of the study The functional diversity of ribonucleic acids (RNAs) is based on their ability to form complex three-dimensional structures. During RNA folding, the formation of off-pathway folding traps often prevents correct folding of RNA. In fact, structural transitions between a single or several different metastable states and the final most-stable state, a process referred to as RNA refolding, constitute the rate-limiting step on the folding pathway towards the functional RNA fold. Proteins with RNA chaperone activity assist in the correct folding and structural rearrangements of RNA molecules by resolving kinetically trapped RNA species in an ATP-independent manner. So far the mode of action by which a strand dissociation is facilitated is not solved. Aim of the study The goal of the study is to characterise the changes in the molecular dynamics of an RNA molecule under the influence of a protein exhibiting RNA chaperone activity. This should result in an answer to the question: "What does an RNA chaperone do to an RNA in order to facilitate its folding?" Thereby we will investigate the structure and dynamics of target RNAs in the absence as well as in the presence of RNA chaperones. As target RNAs two stem loop RNA structures that are involved in functional refolding reactions were chosen. The first RNA element represents the terminator hairpin loop of the trpL gene from E. coli, the second RNA structure is derived from the expression platform of the SAM riboswitch of B. anthracis. The chaperone molecules that will be used in the studies are the StpA protein from E. coli and the ribosomal protein S1 from the same organism, as both molecules were found to exhibit strand displacement activity in a variety of biochemical assays. The change in the protein structure and dynamics of the proteins itself will also be examined as well as the influence of those on an elementary re-folding reaction of the target RNAs. The techniques that will be used include basic biochemical methods but will focus on traditional static as well as modern dynamic NMR methods, such as relaxation experiments, field dependent RDCs and time resolved NMR spectroscopy. The experimental results should lead to the functional and biophysical characterisation of the changes that a chaperone is capable to induce on a dynamic RNA structure. Overall, this includes an entire description of the changes in the thermodynamic states of the molecules and the influence on the kinetics of an RNA refolding reaction.