Elizabeth A. Dethoff

Functional complexity and regulation through RNA dynamics.

Changes to the conformation of coding and non-coding RNAs form the basis of elements of genetic regulation and provide an important source of complexity, which drives many of the fundamental processes of life. Although the structure of RNA is highly flexible, the underlying dynamics of RNA are robust and are limited to transitions between the few conformations that preserve favourable base-pairing and stacking interactions. The mechanisms by which cellular processes harness the intrinsic dynamic behaviour of RNA and use it within functionally productive pathways are complex. The versatile functions and ease by which it is integrated into a wide variety of genetic circuits and biochemical pathways suggests there is a general and fundamental role for RNA dynamics in cellular processes.

Visualizing transient low-populated structures of RNA

The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized so far involve long-lived species that can be captured by structure characterization techniques. Here we report the nuclear magnetic resonance visualization of RNA transitions towards ‘invisible’ excited states (ESs), which exist in too little abundance (2–13%) and for too short a duration (45–250 μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix–junction–helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signalling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.

Characterizing complex dynamics in the transactivation response element apical loop and motional correlations with the bulge by NMR, molecular dynamics, and mutagenesis.

The HIV-1 transactivation response element (TAR) RNA binds a variety of proteins and is a target for developing anti-HIV therapies. TAR has two primary binding sites: a UCU bulge and a CUGGGA apical loop. We used NMR residual dipolar couplings, carbon spin relaxation (R(1) and R(2)), and relaxation dispersion (R(1rho)) in conjunction with molecular dynamics and mutagenesis to characterize the dynamics of the TAR apical loop and investigate previously proposed long-range interactions with the distant bulge. Replacement of the wild-type apical loop with a UUCG loop did not significantly affect the structural dynamics at the bulge, indicating that the apical loop and the bulge act largely as independent dynamical recognition centers. The apical loop undergoes complex dynamics at multiple timescales that are likely important for adaptive recognition: U31 and G33 undergo limited motions, G32 is highly flexible at picosecond-nanosecond timescales, and G34 and C30 form a dynamic Watson-Crick basepair in which G34 and A35 undergo a slow (approximately 30 mus) likely concerted looping in and out motion, with A35 also undergoing large amplitude motions at picosecond-nanosecond timescales. Our study highlights the power of combining NMR, molecular dynamics, and mutagenesis in characterizing RNA dynamics.

Invisible RNA state dynamically couples distant motifs

Significance We recently showed, using NMR relaxation dispersion, that the HIV-1 transactivation response element (TAR) RNA forms a transient state (lifetime ∼45 μs and population ∼13%) through localized changes in base pairing within the apical loop. Here, we report a second transient TAR state that has longer lifetime (∼2 ms) and smaller population (∼0.4%) that simultaneously remodels the bulge and apical loop, which are separated by four Watson–Crick base pairs. This is accomplished by a global change in the register of bulge residues, which pair up with residues in the upper stem, causing reshuffling of base pairs that propagates to the apical loop and creation of three noncanonical base pairs. Thus, transient states provide a mechanism for dynamically coupling distant RNA motifs. Using on- and off-resonance carbon and nitrogen R1ρ NMR relaxation dispersion in concert with mutagenesis and NMR chemical shift fingerprinting, we show that the transactivation response element RNA from the HIV-1 exists in dynamic equilibrium with a transient state that has a lifetime of ∼2 ms and population of ∼0.4%, which simultaneously remodels the structure of a bulge, stem, and apical loop. This is accomplished by a global change in strand register, in which bulge residues pair up with residues in the upper stem, causing a reshuffling of base pairs that propagates to the tip of apical loop, resulting in the creation of three noncanonical base pairs. Our results show that transient states can remodel distant RNA motifs and possibly give rise to mechanisms for rapid long-range communication in RNA that can be harnessed in processes such as cooperative folding and ribonucleoprotein assembly.

Variable helix elongation as a tool to modulate RNA alignment and motional couplings

The application of residual dipolar couplings (RDCs) in studies of RNA structure and dynamics can be complicated by the presence of couplings between collective helix motions and overall alignment and by the inability to modulate overall alignment of the molecule by changing the ordering medium. Here, we show for a 27-nt TAR RNA construct that variable levels of helix elongation can be used to alter both overall alignment and couplings to collective helix motions in a semi-predictable manner. In the absence of elongation, a four base-pair helix II capped by a UUCG apical loop exhibits a higher degree of order compared to a six base-pair helix I (theta(I)/theta(II)=0.56+/-0.1). The principal S(zz) direction is nearly parallel to the axis of helix II but deviates by approximately 40 degrees relative to the axis of helix I. Elongating helix I by three base-pairs equalizes the alignment of the two helices and pushes the RNA into the motional coupling limit such that the two helices have comparable degrees of order (theta(I)/theta(II)=0.92+/-0.04) and orientations relative to S(zz) ( approximately 17 degrees ). Increasing the length of elongation further to 22 base-pairs pushes the RNA into the motional decoupling limit in which helix I dominates alignment (theta(II)/theta(I)=0.45+/-0.05), with S(zz) orientated nearly parallel to its helix axis. Many of these trends can be rationalized using PALES simulations that employ a previously proposed three-state dynamic ensemble of TAR. Our results provide new insights into motional couplings, offer guidelines for assessing their extent, and suggest that variable degrees of helix elongation can allow access to independent sets of RDCs for characterizing RNA structural dynamics.

Furan Approach to Vitamin D Analogues. Synthesis of the A-Ring of Calcitriol and 1α-Hydroxy-3-deoxyvitamin D3

The A-rings of calcitriol (1α,25-dihydroxyvitamin D(3)) and 1α-hydroxy-3-deoxyvitamin D(3) were synthesized using the furan approach. The critical steps in the synthesis of the A-ring of calcitriol involved an asymmetric carbonyl-ene reaction of 3-methylene-2,3-dihydrofuran with 3-(tert-butyldimethylsiloxy)propanal, a diastereoselective Friedel-Crafts hydroxyalkylation, an oxidation of the 2,3-disubstituted furan to give a γ-hydroxybutenolide, and a Peterson olefination. The A-ring (Z)-dienol of calcitriol was synthesized in 12 steps from 3-(tert-butyldimethylsiloxy)propanal in 17% yield.

Packaged and Free Satellite Tobacco Mosaic Virus (STMV) RNA Genomes Adopt Distinct Conformational States

The RNA genomes of viruses likely undergo multiple functionally important conformational changes during their replication cycles, changes that are poorly understood at present. We used two complementary in-solution RNA structure probing strategies (SHAPE-MaP and RING-MaP) to examine the structure of the RNA genome of satellite tobacco mosaic virus inside authentic virions and in a capsid-free state. Both RNA states feature similar three-domain architectures in which each major replicative function-translation, capsid coding, and genome synthesis-fall into distinct domains. There are, however, large conformational differences between the in-virion and capsid-free states, primarily in one arm of the central T domain. These data support a model in which the packaged capsid-bound RNA is constrained in a local high-energy conformation by the native capsid shell. The removal of the viral capsid then allows the RNA genome to relax into a more thermodynamically stable conformation. The RNA architecture of the central T domain thus likely changes during capsid assembly and disassembly and may play a role in genome packaging.

Dynamic Motions of the HIV-1 Frameshift Site RNA

The HIV-1 frameshift site (FS) plays a critical role in viral replication. During translation, the HIV-1 FS transitions from a 3-helix to a 2-helix junction RNA secondary structure. The 2-helix junction structure contains a GGA bulge, and purine-rich bulges are common motifs in RNA secondary structure. Here, we investigate the dynamics of the HIV-1 FS 2-helix junction RNA. Interhelical motions were studied under different ionic conditions using NMR order tensor analysis of residual dipolar couplings. In 150 mM potassium, the RNA adopts a 43°(±4°) interhelical bend angle (β) and displays large amplitude, anisotropic interhelical motions characterized by a 0.52(±0.04) internal generalized degree of order (GDOint) and distinct order tensor asymmetries for its two helices (η = 0.26(±0.04) and 0.5(±0.1)). These motions are effectively quenched by addition of 2 mM magnesium (GDOint = 0.87(±0.06)), which promotes a near-coaxial conformation (β = 15°(±6°)) of the two helices. Base stacking in the bulge was investigated using the fluorescent purine analog 2-aminopurine. These results indicate that magnesium stabilizes extrahelical conformations of the bulge nucleotides, thereby promoting coaxial stacking of helices. These results are highly similar to previous studies of the HIV transactivation response RNA, despite a complete lack of sequence similarity between the two RNAs. Thus, the conformational space of these RNAs is largely determined by the topology of their interhelical junctions.

Pervasive tertiary structure in the dengue virus RNA genome

Significance RNA viruses usurp and reprogram host cells using short RNA genomes. RNA viruses encode the information required for their replication in both their primary sequences and higher-order structures formed when the RNA genome strand folds back on itself, but the extent of higher-order structure has remained unclear. We use a new high-throughput RNA structure probing technology to identify RNA regions with tertiary folds and discover that roughly one-third of the dengue virus RNA genome forms higher-order interactions, many in regions functionally important for replication. This work suggests that tertiary structure elements might be common in large RNAs, and that these regions might contain pockets targetable by small molecules in the design of antiviral therapeutics. RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds—in which helices pack together to form complex 3D structures—and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.