NMR Studies of RNA Conformational Dynamics Induced by Metal Cations and Paromomycin

Many regulatory RNAs adaptively change their conformation on binding to cognate protein and ligand targets or therapeutic molecules but the mechanism by which these conformational changes occur still remains poorly understood. In this thesis we characterize the dynamic properties of two RNA drug targets; HIV-1 TAR RNA and ribosomal A-site rRNA using a battery of NMR experiments that provide information on motions occurring over picosecond to millisecond timescales. We have used residual dipolar couplings (RDCs) in concert with structure-based electrostatic calculations to characterize the dependence of local and inter-helical recognition motions in TAR containing a trinucleotide pyrimidine bulge on the concentration of Na+ and Mg2+. Results revealed that Na+ or Mg2+ induce a similar dynamic transition of TAR from an electrostatic relaxed bent and flexible state to a globally rigid coaxial state, which has a stronger negative charge density and association with counterions. The dynamic transition carries the TAR structure through several of the ligand bound conformations, indicating that metals and electrostatic interactions likely play an important role in adaptive recognition. We used domain-elongation spin relaxation and relaxation dispersion NMR experiments to characterize base flipping and other local motions in the A-site rRNA in the absence and presence of the aminoglycoside antibiotic paromomycin. Our results strongly suggest that A-site can dynamically access conformations in which the two adenines are flipped out at microsecond timescales and that binding to paromomycin is not necessary to induce this transition. These results were then compared to those obtained on a corresponding construct bearing an antibiotic resistance A1408G mutation. This single mutation leads to dramatic changes in the dynamics observed over picosecond to millisecond timescales. We propose that a G-A base-pair reduces the propensity to have both adenine residues looped out, thereby explaining in part the much lower antibiotic affinity for this RNA construct. Taken together, our results provide fundamental new insights into how internal motions occurring on different timescales can drive the conformational changes that accompany molecular recognition.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78756/1/acasiano_1.pd

Constructing RNA dynamical ensembles by combining MD and motionally decoupled NMR RDCs: new insights into RNA dynamics and adaptive ligand recognition

We describe a strategy for constructing atomic resolution dynamical ensembles of RNA molecules, spanning up to millisecond timescales, that combines molecular dynamics (MD) simulations with NMR residual dipolar couplings (RDC) measured in elongated RNA. The ensembles are generated via a Monte Carlo procedure by selecting snap-shot from an MD trajectory that reproduce experimentally measured RDCs. Using this approach, we construct ensembles for two variants of the transactivation response element (TAR) containing three (HIV-1) and two (HIV-2) nucleotide bulges. The HIV-1 TAR ensemble reveals significant mobility in bulge residues C24 and U25 and to a lesser extent U23 and neighboring helical residue A22 that give rise to large amplitude spatially correlated twisting and bending helical motions. Omission of bulge residue C24 in HIV-2 TAR leads to a significant reduction in both the local mobility in and around the bulge and amplitude of inter-helical bending motions. In contrast, twisting motions of the helices remain comparable in amplitude to HIV-1 TAR and spatial correlations between them increase significantly. Comparison of the HIV-1 TAR dynamical ensemble and ligand bound TAR conformations reveals that several features of the binding pocket and global conformation are dynamically preformed, providing support for adaptive recognition via a ‘conformational selection’ type mechanism

Structural characterization of naturally occurring RNA single mismatches

RNA is known to be involved in several cellular processes; however, it is only active when it is folded into its correct 3D conformation. The folding, bending and twisting of an RNA molecule is dependent upon the multitude of canonical and non-canonical secondary structure motifs. These motifs contribute to the structural complexity of RNA but also serve important integral biological functions, such as serving as recognition and binding sites for other biomolecules or small ligands. One of the most prevalent types of RNA secondary structure motifs are single mismatches, which occur when two canonical pairs are separated by a single non-canonical pair. To determine sequence–structure relationships and to identify structural patterns, we have systematically located, annotated and compared all available occurrences of the 30 most frequently occurring single mismatch-nearest neighbor sequence combinations found in experimentally determined 3D structures of RNA-containing molecules deposited into the Protein Data Bank. Hydrogen bonding, stacking and interaction of nucleotide edges for the mismatched and nearest neighbor base pairs are described and compared, allowing for the identification of several structural patterns. Such a database and comparison will allow researchers to gain insight into the structural features of unstudied sequences and to quickly look-up studied sequences

Review NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings

An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large-amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (<milliseconds). Here, we review RDC methodology for studying RNA structural dynamics and survey what has been learnt thus far from application of these methods. Future methodological challenges are also identified. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 384–402, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56044/1/20765_ftp.pd

NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5′ end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn2+ bound to a P4 helix mimic is six-coordinate, with an average Zn-O/N bond distance of 2.08 Å. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn2+ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new “metal cocktail” chemical shift perturbation strategy involving titrations with , Zn2+, and confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution