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Noncoding 7SK snRNA is believed to play an important role in the recruitment of P-TEFb by viral protein Tat to stimulate HIV processive transcription. Because HIV-2 TAR RNA and 7SK both evolved to feature a dinucleotide bulge region, compared to the trinucleotide bulge for HIV-1 TAR, ultrafast time-resolved fluorescence spectroscopy has been used to probe the conformational landscape of HIV-2 TAR and 7SK-SL4 RNA to monitor the conformational changes upon Tat binding. Our studies demonstrate that both HIV-1/2 TAR and 7SK-SL4 sample heterogeneous ensembles in the free state and undergo distinct conformational transitions upon Tat binding. These findings provide exquisite knowledge on the conformational complexity and intricate mechanism of molecular recognition and pave the way for drug design and discovery that incorporate dynamics information.

Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.

RNA conformational dynamics and the resulting structural heterogeneity play an important role in RNA functions, e.g., recognition. Recognition of HIV-1 TAR RNA has been proposed to occur via a conformational capture mechanism. Here, using ultrafast time-resolved fluorescence spectroscopy, we have probed the complexity of the conformational landscape of HIV-1 TAR RNA and monitored the position-dependent changes in the landscape upon binding of a Tat protein-derived peptide and neomycin B. In the ligand-free state, the TAR RNA samples multiple families of conformations with various degrees of base stacking around the three-nucleotide bulge region. Some subpopulations partially resemble those ligand-bound states, but the coaxially stacked state is below the detection limit. When Tat or neomycin B binds, the bulge region as an ensemble undergoes a conformational transition in a position-dependent manner. Tat and neomycin B induce mutually exclusive changes in the TAR RNA underlying the mechanism of allosteric inhibition at an ensemble level with residue-specific details. Time-resolved anisotropy decay measurements revealed picosecond motions of bases in both ligand-free and ligand-bound states. Mutation of a base pair at the bulge--stem junction has differential effects on the conformational distributions of the bulge bases. A dynamic model of the ensemble view of the conformational landscape for HIV-1 TAR RNA is proposed, and the implication of the general mechanism of RNA recognition and its impact on RNA-based therapeutics are discussed.

Nosiheptide-resistance methyltransferase (NHR) of Streptomyces actuosus is a class IV methyltransferase of the SpoU family and methylates 23S rRNA at nucleotide adenosine corresponding to A1067 in Escherichia coli. Such methylation is essential for resistance against nosiheptide, a sulfur peptide antibiotic, which is produced by the nosiheptide-producing strain, S. actuosus. Here, we report the crystal structures of NHR and NHR in complex with SAM (S-adenosyl-l-methionine) at 2.0 and 2.1 A resolution, respectively. NHR forms a functional homodimer, and dimerization is required for methyltransferase activity. The monomeric NHR is comprised of the N-terminal RNA binding domain (NTD) and the C-terminal catalytic domain (CTD). Overall, the structure of NHR suggests that the methyltransferase activity is achieved by "reading" the RNA substrate with NTD and "adding" methyl group using CTD. Comprehensive mutagenesis and methyltransferase activity assays reveal critical regions for SAM binding in CTD and loops (L1 and L3) essential for RNA recognition in NTD. Finally, the catalytic mechanism and structural model that NHR recognizes 23S rRNA is proposed based on the structural and biochemical analyses. Thus, our systematic structural studies reveal the substrate recognition and modification by the nosiheptide-resistance methyltransferase.

RNAs often exhibit a high degree of conformational dynamics and heterogeneity, leading to a rugged energy landscape. However, the roles of conformational heterogeneity and rapid dynamics in molecular recognition or RNA function have not been extensively elucidated. Ultrafast time-resolved fluorescence spectroscopic experiments were used here to probe picosecond dynamics of the theophylline-binding RNA aptamer. These studies showed that multiple conformations are populated in the free RNA indicating that this aptamer employs a conformational capture mechanism for ligand binding. The base on residue 27 in an internal loop exists in at least three conformational states in the free RNA, including binding competent and incompetent states that have distinct fluorescence decay signatures indicating different base stacking interactions. Picosecond dynamics were also detected by anisotropy experiments, where these motions indicate additional dynamics for base 27. The picosecond data show that theophylline binding shifts the equilibrium for conformations of base 27 from primarily stacked in the free RNA to mostly unstacked in the RNA-theophylline complex, as observed in the previous NMR structure. In contrast, base 10 in a second internal loop is mostly pre-organized in the free RNA, consistent with it being stacked between G11 and G25, as is observed in the bound state. Picosecond dynamics were also measured on a modified aptamer that binds with higher affinity to 3-methylxanthine than theophylline. The modified aptamer shows less heterogeneity in the aptamer-3-methylxanthine complex than what is observed in the theophylline aptamer-theophylline complex.

Efforts toward the development of RNA-based drug leads have been challenging because of the complexity and dynamic nature of RNA structures as therapeutic targets. The transactivation response (TAR) RNA and cognate Tat protein of HIV have long been recognized as promising antiviral targets, and recent works have identified potentially potent inhibitors of the viral RNA-protein interaction. A new class of such inhibitors, conformationally constrained cyclic peptide mimetics of Tat, has been demonstrated to inhibit the HIV life cycle. We have previously probed the complexity and dynamics of TAR RNAs in their free states, as well as conformational shifting by various peptide and small molecule ligands. In this work, we have used an ultrafast dynamics approach to probe the interactions between TAR RNAs and one of the representatives of cyclic peptide inhibitors, L22. Our studies demonstrated that cyclic L22 specifically recognizes TAR RNAs with a unique single binding site compared to two binding sites for linear Tat protein. Although both Tat and L22 bind to the TAR RNAs as a β-hairpin structure, cyclization in L22 allows it to be a more efficient ligand from a population shifting perspective. This study provided unique insights into drug design with desired properties to differentiate similar structures based on distinct dynamic behaviors.

The dynamic behavior of the rRNA A-site plays an important functional role. We have employed femtosecond time-resolved spectroscopy to investigate the nature of the conformational dynamics. In the drug-free state, the A-site samples multiple distinct conformations. Drug binding shifts the population distribution in a drug-specific manner. Motions of bases on nanosecond and picosecond time scales are differentially affected by the drug binding. Our results underscore the importance of understanding the detailed dynamic picture of molecular recognition by resolving dynamics in the distinct picosecond time regime and facilitate development of antimicrobial drugs targeting dynamic RNAs.

High-resolution crystal structures and biophysical analyses of purine-sensing riboswitches have revealed that a network of hydrogen bonding interactions appear to be largey responsible for discrimination of cognate ligands against structurally related compounds. Here we report that by using femtosecond time-resolved fluorescence spectroscopy to capture the ultrafast decay dynamics of the 2-aminopurine base as the ligand, we have detected the presence of multiple conformations of the ligand within the binding pockets of one guanine-sensing and two adenine-sensing riboswitches. All three riboswitches have similar conformational distributions of the ligand-bound state. The known crystal structures represent the global minimum that accounts for 50-60% of the population, where there is no significant stacking interaction between the ligand and bases of the binding pocket, but the hydrogen-bonding cage collectively provides an electronic environment that promotes an ultrafast ( approximately 1 ps) charge transfer pathway. The ligand also samples multiple conformations in which it significantly stacks with either the adenine or the uracil bases of the A21-U75 and A52-U22 base pairs that form the ceiling and floor of the binding pocket, respectively, but favors the larger adenine bases. These alternative conformations with well-defined base stacking interactions are approximately 1-1.5 kcal/mol higher in DeltaG degrees than the global minimum and have distinct charge transfer dynamics within the picosecond to nanosecond time regime. Inside the pocket, the purine ligand undergoes dynamic motion on the low nanosecond time scale, sampling the multiple conformations based on time-resolved anisotropy decay dynamics. These results allowed a description of the energy landscape of the bound ligand with intricate details and demonstrated the elastic nature of the ligand recognition mode by the purine-sensing riboswitches, where there is a dynamic balance between hydrogen bonding and base stacking interactions, yielding the high affinity and specificity by the aptamer domain.

The dynamic nature of ribozymes represents a significant challenge in elucidating their structure−dynamics−function relationship. Here, using femtosecond time-resolved spectroscopy and other biophysical tools, we demonstrate that the active site of leadzyme does not have a unique structure, but rather samples an ensemble of conformations that undergo picosecond structural changes. Various base modifications have a profound context-dependent impact on the catalysis.

Unpaired bases at the end of an RNA duplex (dangling ends) can stabilize the core duplex in a sequence-dependent manner and are important determinants of RNA folding, recognition, and functions. Using 2-aminopurine as a dangling end purine base, we have employed femtosecond time-resolved fluorescence spectroscopy, combined with UV optical melting, to quantitatively investigate the physical and structural nature of the stacking interactions between the dangling end bases and the terminal base pairs. A 3'-dangling purine base has a large subpopulation that stacks on the guanine base of the terminal GC or UG pair, either intrastrand or cross-strand depending on the orientation of the pair, thus providing stabilization of different magnitudes. On the contrary, a 5'-dangling purine base only has a marginal subpopulation that stacks on the purine of the same strand (intrastrand) but has little cross-strand stacking. Thus a 5'-dangling purine does not provide significant stabilization. These stacking structures are not static, and a dangling end base samples a range of stacked and unstacked conformations with respect to the terminal base pair. Femtosecond time-resolved anisotropy decay reveals certain hindered base conformational dynamics that occur on the picosecond to nanosecond time scales, which allow the dangling base to sample these substates. When the dangling purine is opposite to a U and is able to form a potential base pair at the end of the duplex, there is an interplay of base stacking and hydrogen-bonding interactions that depends on the orientation of the base pair relative to the adjacent GC pair. By resolving these populations that are dynamically exchanging on fast time scales, we elucidated the correlation between dynamic conformational distributions and thermodynamic stability.

The orientation of the myosin neck was monitored using electron paramagnetic resonance (EPR) spectroscopy. Gizzard regulatory light chain was labeled with a nitroxide spin probe and exchanged for the native subunit, located in the myosin neck, in rabbit psoas muscle fibers. The EPR spectra of rigor fibers indicated a substantial degree of probe immobilization and showed a strong dependence on the orientation of the fiber axis relative to the magnetic field, indicating that the neck was ordered in this state. Spectra of relaxed fibers at 24 degrees C showed that the neck was disordered, but the spectra of relaxed fibers at 4 degrees C indicated that the neck was partially ordered. Active fibers at the two temperatures produced spectra identical to relaxed fibers, indicating that no novel angles could be seen in the neck during the powerstroke. Proteolytic fragments of myosin, S1 and HMM, were exchanged with labeled light chains and bound to thin filaments in unlabeled fibers. The distribution of probe orientations for HMM was identical to that of labeled rigor fibers, while S1 showed a slightly different distribution, suggesting that the neck is distorted (by a few degrees) by the interactions of the two heads of myosin when bound to actin.