Non-specific binding of Na + and Mg 2+ to RNA determined by force spectroscopy methods

Bizarro, Cristiano Valim; Alemany, Anna; Ritort, Fèlix

doi:10.1093/nar/gks289

Cited by 80 publications

(126 citation statements)

References 93 publications

(226 reference statements)

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“…For both ppp-15-15-15 and ppp-0-15-0, which lacks single-stranded tails, the binding affinities decrease strongly in AU60/5 Mg (Supplemental Table S4). This observation is consistent with previous studies indicating that Mg 2+ is ∼100-fold more efficient at charge screening for RNAs compared to monovalent cations (Bizarro et al 2012;Chen et al 2012). As indicated by a similar reduction in the maximum RP 2 for the two RNAs, there are no specific Mg 2+ effects conferred by the single-stranded tails.…”

Section: Effect Of Tail Deletionssupporting

confidence: 92%

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Mayo

Wong

Lopez

et al. 2016

RNA

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Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem-loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5 ′ -triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5 ′ -and 3 ′ -single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.

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Section: Effect Of Tail Deletionssupporting

confidence: 92%

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Mayo

Wong

Lopez

et al. 2016

RNA

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“…A reduction of the hairpin unfolding free-energy barrier may be caused by increasing concentrations of NC or by decreasing the concentration of salt in solution. Previous investigations have examined effects on hairpin force-unfolding due to changing solution ionic strength (22,28), temperature (29), and stabilizing ligand (30). Importantly, in all of these previous studies the location of the opening transition state was not affected by the variation of solution conditions or the addition of ligand.…”

Section: Specific Binding Drives the Unusual Shift In The Tar Transitmentioning

confidence: 99%

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

McCauley

Rouzina

Manthei

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA-DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA-DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.single molecule | force spectroscopy | RNA stretching | RNA binding T he transactivation response (TAR) RNA hairpin is a 59-nt sequence in the long-terminal repeat (LTR) of the HIV-1 genome that forms a 24-bp hairpin ( Fig. 1A) (1). This structure is essential in promoting viral transactivator protein (Tat)-mediated transcription. The protein-RNA complex further enhances LTR promoter activity (2). The highly stable TAR hairpin structure that stimulates viral RNA transcription becomes a liability during the early stage of a new infection, as TAR hairpins inhibit the minus-strand transfer step required for reverse transcription (1). To alleviate this inhibition, successful reverse transcription requires a key viral chaperone, the nucleocapsid (NC) protein. In vitro experiments have shown a 3,000-fold stimulation of the rate-limiting step of minus-strand transfer in the presence of NC (3), as NC is required to destabilize TAR RNA and the complementary repeat TAR DNA hairpin to allow subsequent strand annealing (1).HIV-1 NC is only 55 aa long, consisting of two highly conserved CCHC zinc fingers and a basic N terminus (1) (Fig. 1B). The multiple roles of NC during reverse transcription all use the same "chaperone" activity (1), which describes HIV-1 NC's ability to facilitate the rearrangement of nucleic acids into the most stable structures, with the lowest free energy (1). This chaperone activity is characterized by nucleic acid aggregation, duplex destabilization, and rapid kinetics of protein-nucleic acid interactions (3, 4). Aromatic residues in...

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“…Little is known about whether nucleic acid sequences undergoing replication, transcription and/or translation might also need special adaptations to function in organisms using a salt-in strategy for osmotic balance. Nucleic acid structure and stability are known to be exquisitely sensitive to ionic concentrations in vitro, especially magnesium, which is orders of magnitude more active than sodium or potassium in increasing double-stranded DNA melting temperature (Hartwig, 2001;Owczarzy et al, 2008) and stabilizing RNA hairpin structures (Bizarro et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Seasonal fluctuations in ionic concentrations drive microbial succession in a hypersaline lake community

et al. 2013

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Microbial community succession was examined over a two-year period using spatially and temporally coordinated water chemistry measurements, metagenomic sequencing, phylogenetic binning and de novo metagenomic assembly in the extreme hypersaline habitat of Lake Tyrrell, Victoria, Australia. Relative abundances of Haloquadratum-related sequences were positively correlated with co-varying concentrations of potassium, magnesium and sulfate, but not sodium, chloride or calcium ions, while relative abundances of Halorubrum, Haloarcula, Halonotius, Halobaculum and Salinibacter-related sequences correlated negatively with Haloquadratum and these same ionic factors. Nanohaloarchaea and Halorhabdus-related sequence abundances were inversely correlated with each other, but not other taxonomic groups. These data, along with predicted gene functions from nearly-complete assembled population metagenomes, suggest different ecological phenotypes for Nanohaloarchaea and Halorhabdus-related strains versus other community members. Nucleotide percent G þ C compositions were consistently lower in community metagenomic reads from summer versus winter samples. The same seasonal G þ C trends were observed within taxonomically binned read subsets from each of seven different genus-level archaeal groups. Relative seasonal abundances were also linked to percent G þ C for assembled population genomes. Together, these data suggest that extreme ionic conditions may exert selective pressure on archaeal populations at the level of genomic nucleotide composition, thus contributing to seasonal successional processes. Despite the unavailability of cultured representatives for most of the organisms identified in this study, effective coordination of physical and biological measurements has enabled discovery and quantification of unexpected taxon-specific, environmentally mediated factors influencing microbial community structure.

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Non-specific binding of Na + and Mg 2+ to RNA determined by force spectroscopy methods

Cited by 80 publications

References 93 publications

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

Seasonal fluctuations in ionic concentrations drive microbial succession in a hypersaline lake community

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