Invisible RNA state dynamically couples distant motifs

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

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

Structural and Dynamic Basis for Assembly of the HIV-1 TAR-Tat Ribonucleoprotein Complex

The transactivation response element (TAR), located at the 5' end of the HIV-1 genome, regulates the transcription elongation step of viral RNA. The TAR stem-loop binds the HIV viral transactivator protein (Tat), which enhances viral transcription by recruiting the positive transcription elongation factor b (P-TEFb). The TAR-Tat interaction and formation of the TAR/Tat/P-TEFb ribonucleoprotein (RNP) complex remains poorly understood from a structural and dynamic standpoint. To better understand the formation of this complex, we have studied the structural and dynamic features of free TAR to elucidate motions in both the bulge and apical loop that may be important for adaptive recognition of protein targets and, ultimately, for anti-viral therapeutic design. We used a combination of nuclear magnetic resonance (NMR), molecular dynamics (MD), and mutagenesis to characterize the structural dynamics of free TAR. Residual dipolar couplings (RDCs), motionally-decoupled 13C relaxation (R1, R2), and 13C R1ρ relaxation dispersion were used in combination to characterize global and local motions occurring on fast (ps to ns) and slow (us to ms) timescales. These results were compared to an MD simulation of TAR. Our results show that the apical loop and bulge act as independent dynamical centers, with the apical loop undergoing complex dynamics on the microsecond to picosecond timescale that may be important for adaptive recognition. We have characterized a lowly-populated structure of the loop that is otherwise “invisible” to traditional NMR techniques, representing the first-ever characterization of an “invisible” state of an RNA loop by NMR. Overall, our results establish a broadly useful approach for characterizing the dynamics of RNA loops and shed light on the relationship between dynamics and biological function for the TAR element in the HIV genome.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86484/1/dethoffe_1.pd

Invisible RNA state dynamically couples distant 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

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

Kinetic Resolution of the Atomic 3D Structures Formed by Ground and Excited Conformational States in an RNA Dynamic Ensemble

Knowing the 3D structures formed by the various conformations populating the RNA free-energy landscape, their relative abundance, and kinetic interconversion rates is required to obtain a quantitative and predictive understanding of how RNAs fold and function at the atomic level. While methods integrating ensemble-averaged experimental data with computational modeling are helping define the most abundant conformations in RNA ensembles, elucidating their kinetic rates of interconversion and determining the 3D structures of sparsely populated short-lived RNA excited conformational states (ESs) remains challenging. Here, we developed an approach integrating Rosetta-FARFAR RNA structure prediction with NMR residual dipolar couplings and relaxation dispersion that simultaneously determines the 3D structures formed by the ground-state (GS) and ES subensembles, their relative abundance, and kinetic rates of interconversion. The approach is demonstrated on HIV-1 TAR, whose six-nucleotide apical loop was previously shown to form a sparsely populated (∼13%) short-lived (lifetime ∼ 45 μs) ES. In the GS, the apical loop forms a broad distribution of open conformations interconverting on the pico-to-nanosecond time scale. Most residues are unpaired and preorganized to bind the Tat-superelongation protein complex. The apical loop zips up in the ES, forming a narrow distribution of closed conformations, which sequester critical residues required for protein recognition. Our work introduces an approach for determining the 3D ensemble models formed by sparsely populated RNA conformational states, provides a rare atomic view of an RNA ES, and kinetically resolves the atomic 3D structures of RNA conformational substates, interchanging on time scales spanning 6 orders of magnitude, from picoseconds to microseconds

Kinetic Resolution of the Atomic 3D Structures Formed by Ground and Excited Conformational States in an RNA Dynamic Ensemble

Knowing the 3D structures formed by the various conformations populating the RNA free-energy landscape, their relative abundance, and kinetic interconversion rates is required to obtain a quantitative and predictive understanding of how RNAs fold and function at the atomic level. While methods integrating ensemble-averaged experimental data with computational modeling are helping define the most abundant conformations in RNA ensembles, elucidating their kinetic rates of interconversion and determining the 3D structures of sparsely populated short-lived RNA excited conformational states (ESs) remains challenging. Here, we developed an approach integrating Rosetta-FARFAR RNA structure prediction with NMR residual dipolar couplings and relaxation dispersion that simultaneously determines the 3D structures formed by the ground-state (GS) and ES subensembles, their relative abundance, and kinetic rates of interconversion. The approach is demonstrated on HIV-1 TAR, whose six-nucleotide apical loop was previously shown to form a sparsely populated (∼13%) short-lived (lifetime ∼ 45 μs) ES. In the GS, the apical loop forms a broad distribution of open conformations interconverting on the pico-to-nanosecond time scale. Most residues are unpaired and preorganized to bind the Tat-superelongation protein complex. The apical loop zips up in the ES, forming a narrow distribution of closed conformations, which sequester critical residues required for protein recognition. Our work introduces an approach for determining the 3D ensemble models formed by sparsely populated RNA conformational states, provides a rare atomic view of an RNA ES, and kinetically resolves the atomic 3D structures of RNA conformational substates, interchanging on time scales spanning 6 orders of magnitude, from picoseconds to microseconds