The function of virtually every regulatory RNA relies on molecular flexibility that allows it to fold into different conformations required during the course of assembly, catalysis, and regulation. Though NMR spectroscopy has proven to be one of the most powerful techniques for probing internal dynamics in proteins uniquely providing site-specific information, existing paradigms break down when applied to nucleic acids due to their unique spin physics and motional properties. The objective of this CAREER project is to develop and apply novel NMR methods for characterizing RNA dynamics from picosecond-to-millisecond timescales. At the heart of these methods is a domain elongation strategy that provides a means for interpreting spin relaxation data and residual dipolar couplings in terms of site-specific dynamical parameters. These methods will be generalized to allow studies of larger RNAs and used to investigate the network of internal motional modes that drives conformational adaptation in the transactivation response element (TAR) RNA upon recognition of cognate targets. This will then be followed by dynamic studies of a larger (80 nt), more complex thiamine pyrophosphate (TPP) riboswitch RNA domain from E. coli mRNA which represents a new class of regulatory RNAs that regulate gene expression by switching conformation upon target recognition. These investigations will yield the first site-specific characterization of internal motions in RNA providing fundamental new insight into how structure and dynamics come together to create function. By deepening our understanding of the physical principles governing RNA functional dynamics, this research will also benefit applications seeking to rationally control RNA activity, including the design of RNA sensors. The methodology can easily be adapted to study dynamics in DNA molecules.
The main objective of the educational component is to foster greater awareness within the community about the excitement that surrounds scientific research and discovery. To this end, a one-day forum will be organized once a year at the University of Michigan that is open to the community and free of charge that presents and celebrates several major discoveries specifically as they pertain to the PI's research field of structural biology. From Linus Pauling's characterization of the chemical bond to the story surrounding the discovery of the DNA double helix, the forum will strive to explain in laymen's terms the intellectual strides that led to the discovery, and explore the human, historical, and cultural stories behind these achievements. The forum will include lectures followed by tours of state-of-the-art facilities used in biophysics research. The forum will be summarized in a pedagogical handbook which will be made available to the public via the internet. The forum is expected to enhance existing interactions between the Department of Chemistry and Biophysics Research Division at the University of Michigan and middle and high school students and teachers in the Ann Arbor and surrounding areas. In addition to one graduate student, this research project will be carried out by two undergraduate students currently in the PI's laboratory. This will help sustain a strong tradition of training undergraduate students in the PI's laboratory. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Experimental Physical Chemistry Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.
Project Outcome The overall objective of this proposal was to develop and apply novel NMR methods for characterizing the dynamic properties of large RNA and DNA molecules at atomic resolution with the goal of elucidating how these structures change adaptively to carry out their biological functions. The goal was to understand the role of dynamics in biological important conformational changes that take place in viral and bacterial RNA elements. During the course of the project, we also applied the newly developed methods to DNA, culminating in an important discovery regarding the nature of the iconic DNA double helix. AIM #1 Characterize the variability of nucleobase carbon CSAs across different RNA structural environments. The interpretation of NMR data in terms of dynamics often requires accurate knowledge regarding the chemical shift anisotropies of targeted nuclei. In our studies of TAR and the TAR-ARG complex, we have shown that the expected variability in the carbon CSAs in non-canonical residues will not adversely affect quantitative analysis of relaxation and RCSA data particularly for highly flexible non-canonical residues of interest. This diminishes the need to more fully characterize the CSA variability across different contexts. AIM #2 Develop and apply 13C relaxation methods to characterize local and collective motions in TAR at picosecond-to-nanosecond timescales. We developed NMR techniques that make it possible to characterize motions in large nucleic acids taking place at picosecond to nanosecond timescales. These experiments showed that RNA sites that undergo changes on binding to partners are often flexible, and that binding can lead to both stabilization of structure and increased dynamics at certain sites. We then tangentially developed a new type of NMR experiment – referred to as low spin lock field R1rho experiments for characterizing even slower motions in nucleic acids. With this new experiment we showed that DNA and RNA alike transiently morph into distinct conformations that play biologically important roles. For example, we showed that Watson-Crick base-pairs flip to adopt Hoogsteen base-pairs that define a new layer of genetic information. AIM #3 Apply RDC methods to characterize collective motions in TAR at picosecond-to- millisecond timescales. Obtain ‘structural’ evidence for TAR adaptation through tertiary structure capture. We have developed NMR methods for visualizing – in 3D- motions of helices in RNA molecules. Results showed that HIV-1 TAR RNA dynamically samples all of its ligand bound conformations and that binding most likely proceeds by having ligands capture pre-existing conformations. With these methods, we have also observed fraying motions at the ends of DNA that penetrate deep (7 base-pairs) within the DNA helix and gradually fade away towards the helix interior. AIM #4 Dynamics in the TPP riboswitches: How do riboswitches dynamically sense their targets? We have used our NMR as well as computational methods (in collaboration with the Charlie Brooks lab at the University of Michigan) tocharacterize the dynamic properties of a single-stranded RNA tail derived from the PreQ1 RNA riboswitch – a prototypical example of an RNA molecule that switches conformation in response to cellular signals. Our studies showed that the PreQ single strand pre-adopts a single stranded helical structure and that this pre-ordering may be important to facilitate ligand binding and docking of the single strand onto an apical loop Education Activity The main objective of the educational component was to foster greater awareness within the community about the excitement that surrounds scientific research and discovery. Our goal is to engage a wide spectrum of the community and to enhance the basic learning experience normally received in the classroom by focusing not only on the basic underlying concepts but also on the process of inquiry-driven investigation. We sought to meet this objective by developing two new courses: 1. "Biophysics 120 –The hidden mysteries of the double helix" ins a freshman seminar which focuses on scientific discoveries in the life science particularly the discovery of the DNA double helix and emergence of structural biology. The course emphasized the "human experience" in the major discoveries that ultimately led to our modern understanding of genes and the birth of structural biology as a field. 2. Biophysics 440 –"Biophysics of Diseases" an upper level undergraduate course which deconstructs current and emerging diseases in terms of the malfunctioning of nucleic acids, proteins, and membranes and interactions between them. The diseases covered will include Alzheimer’s, Parkinson’s, Creutzfeldt-Jakob disease (or Mad-Cow disease), HIV, a variety of bacterial infections, and other biological disorders. The course emphasized how a basic biophysical understanding of diseases can guide the rational design of therapeutics and build further the excitement that surrounds scientific discovery in the context of fighting diseases. The above two courses have now been fully integrated in a new University of Michigan undergraduate Biophysics major.
