Mechanism of the RNA chaperone RocC

Numerous biological processes are regulated by molecular chaperones, which promote the correct formation of biomolecular structure. Ribonucleic acid (RNA) chaperones play a particularly intriguing role by regulating the transfer of genetic information into proteins. The underlying molecular processes are inherently dynamic in nature and rely on the participating biomolecules to flexibly adjust their three-dimensional structures to changing requirements. Indeed, it has been recognized that the structural flexibility of these biomolecules appears to be essential and necessary for biological function. While standard methods for structure determination can be used to obtain predominantly static information about biomolecules, dynamic nuclear magnetic resonance (NMR) spectroscopy provides experimental means to identify and characterize structural flexibility at atomic resolution. In this stand-alone project of the FWF we will use dynamic NMR spectroscopy to provide a comprehensive description of chaperoning in the RocC-RocR system. The RNA chaperone RocC regulates bacterial gene expression by binding to the small non-coding RNA molecule RocR. In turn, RocR recognizes and binds to a complementary sequence in messenger RNA (mRNA) for regulation. Within this scheme, the protein RocC acts as a chaperone by promoting the formation base pairs between RocR and its target mRNA. To date, it is not known how exactly chaperoning occurs and what the functional role of structural flexibility in this process might be. Dynamic NMR spectroscopy will be used to directly monitor structural flexibility of the chaperone RocC and its interaction partner RocR. We will probe whether and how chaperone flexibility is transferred from RocC to RocR upon binding, which is probably required for efficient recognition and binding of the target mRNA molecule. Low-populated conformers, which can be transiently formed by flexible molecules, will be characterized in detail. Using site-specific isotope labeling of RocC and its interaction partner RocR, NMR experiments will be implemented that provide insight into structural flexibility in a quantitative manner. By integration of orthogonal techniques we will be able to observe both biomolecular components of the system, chaperone and RNA. This will establish a quantitative description of the interplay between structure, flexibility and binding, and create a basis for understanding how chaperoning works in detail.