Conformational Flexibility of Four-way Junctions in RNA

Hohng, Sungchul; Wilson, Timothy J.; Tan, Elliot; Clegg, Robert M.; Lilley, David M.J.; Ha, Taekjip

doi:10.1016/j.jmb.2003.12.014

Cited by 82 publications

(83 citation statements)

References 31 publications

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“…The four-way junction in the Hairpin ribozyme, an example of a HJH motif, positions its adjoining helices into biased configurations, specifically guiding complementary regions involved in tertiary interactions into close proximity (Walter et al 1998;Tan et al 2003;Hohng et al 2004); changing the topology of its junction (e.g., changing a four-way junction to a three-way, double-stranded, or single-stranded junction) dramatically alters its stability and dynamics (Walter et al 1999;Zhuang et al 2002;Tan et al 2003). Simply mutating the junction sequence in the independently folding P4-P6 subdomain of the Tetrahymena ribozyme can adversely affect its folding stability (Szewczak and Cech 1997), and altering junction length has been shown to affect the catalytic efficiency of simple self-aminoacylating ribozymes (Lehmann et al 2007).…”

Section: Discussionmentioning

confidence: 99%

Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

Chu¹,

Lipfert²,

Bai³

et al. 2009

RNA

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Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA's nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single-or double-stranded polyethylene glycol tethers, whose lengths correspond to those typically observed in junctions from structured RNAs. To dissect the thermodynamic properties of these simple motifs, we computationally probed how junction topology, electrostatics, and tertiary contact location influenced folding stability. Small-angle X-ray scattering was used to assess our predictions. Single-or double-stranded junctions, independent of sequence, greatly reduce the space of allowed helical conformations and influencing the preferred location and orientation of their adjoining helices. A double-stranded junction guides the helices along a hinge-like pathway. In contrast, a single-stranded junction samples a broader set of conformations and has different preferences than the double-stranded junction. In turn, these preferences determine the stability and distinct specificities of tertiary structure formation. These sequence-independent effects suggest that properties as simple as a junction's topology can generally define the accessible conformational space, thereby stabilizing desired structures and assisting in discriminating against misfolded structures. Thus, junction topology provides a fundamental strategy for transcending the limitations imposed by the low information content of RNA primary sequence.

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Section: Discussionmentioning

confidence: 99%

Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

Chu¹,

Lipfert²,

Bai³

et al. 2009

RNA

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“…According to this perspective, HJH elements determine the conformational ensemble explored by unfolded RNA, and ensembles have been demonstrated for highly conserved junctions such as the kink-turn and four-way junction and the biologically important trans-activation response element RNA junction (29)(30)(31). From this perspective, the probability of forming tertiary interactions depends on the fraction of time that the ensemble aligns two tertiary elements such that contacts form between them.…”

Section: Significancementioning

confidence: 99%

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Bisaria

Greenfeld

Limouse

et al. 2017

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

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Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔG align , the probability of aligning tertiary contact partners, and ΔG tert , the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔG HJH ) or from changes in the electrostatic environment (ΔG +/− ) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔG tert ). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔG tert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.RNA folding | single-molecule FRET | RNA tertiary structure | folding kinetics | folding thermodynamics S tructured RNAs are integral to many biological processes, including translation, genome maintenance, and the regulation of gene expression (1, 2). These processes require RNA to fold into intricate 3D structures and to undergo a series of structural transitions (3, 4). As such, an important goal has been to characterize these states, in terms of their conformations and the rates and equilibria that describe the transitions between them (5-8). A more distant but ultimately far-reaching challenge is to quantitatively predict the kinetics and thermodynamics of RNA transitions, an accomplishment that would demonstrate fundamental understanding and effectuate engineering of these systems.Quantitative analyses of the melting temperatures for nucleic acid duplexes led to predictive rules for RNA secondary structure stability, known as nearest neighbor or Turner rules, such that the energetics of helix formation can be predicted by considering only the identity of each base pair, the identity of its immediate neighbors, and the salt concentration (9-11). In this model, the energetic contribution of each base pair step is additive and can be summed to predict the free energy to a...

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“…Molecules were excited by TIR of a 532 nm laser (and a 633 nm laser for alternate excitation) and fluorescence was measured with 100 ms time resolution. Annealed biotin modified enzyme-substrate complex (1 µM Enzyme + 2 µM substrate) was diluted and immobilized on a BSA-biotin-Neutravidin (Pierce) coated quartz slide via Neutravidin-biotin interactions 38 . The DNA concentration was adjusted until a sufficient number of molecules were observed (usually 100-200 pM).…”

Section: Smfret Studymentioning

confidence: 99%

Dissecting metal ion–dependent folding and catalysis of a single DNAzyme

et al. 2007

Self Cite

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Protein metalloenzymes use various modes for functions where metal-dependent global conformational change is required in some cases, but not in others. In contrast, most ribozymes appear to require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using single molecule fluorescence resonance energy transfer (FRET). Addition of Zn 2+ and Mg 2+ resulted in a folding step followed by cleavage reaction, suggesting that the DNAzyme may require metaldependent global folding for activation. In the presence of Pb 2+ , however, cleavage reaction occurred without a precedent folding step, suggesting that the DNAzyme may be prearranged to accept Pb 2+ for the activity. This feature may contribute to the remarkably fast Pb 2+ -dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.Metal ion-dependent folding can play a critical role in metalloenzyme function. Understanding the relationship between folding and reaction is important in obtaining deeper insight into the enzyme mechanism. For protein metalloenzymes, an active-site metal-dependent global folding precedes enzymatic reaction in some cases while such a folding is not required in others 1 . These different modes of activation may fulfill different functions. For example, a reaction preceded by a folding step may be responsible for an allosteric effect in many enzyme functions, or such a folding step may contribute to the overall reaction. Competing Financial Interests StatementThe authors declare there are no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptIn the early 1980s, RNA molecules that can catalyze enzymatic reactions were discovered and named ribozymes 2,3 . This discovery was then followed by demonstrations in the 1990s that DNA can also act as enzymes, termed deoxyribozymes or DNAzymes 4-7 . With only four nucleotides as building blocks versus twenty in proteins, nucleic acid enzymes may need to recruit cofactors to perform some functions. Metal ions are a natural choice and indeed most nucleic acid enzymes require metal ions for function under physiological conditions and therefore, are metalloenzymes. Even though DNAzymes constitute the newest metalloenzyme family, they have already been used in a number of applications such as therapeutic agents 8 , biosensors [9][10][11][12] , and nanomaterials assembly 13 , often because DNAzymes are more stable against hydrolysis and more cost-effective to produce than proteins or ribozymes. A primary example is the 8-17 DNAzyme which cleaves a DNA substrate containing one RNA base at the cleavage site (Fig. 1a) Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript DNAzyme constructsThe original 8-17 DNAzyme was labeled with a fluorophore (Alexa fluoro 488) and two quenchers (Dabcyl) for bulk cleavage activity assays (Fig. 1a). To carry out the smFRET...

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Conformational Flexibility of Four-way Junctions in RNA

Cited by 82 publications

References 31 publications

Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

Dissecting metal ion–dependent folding and catalysis of a single DNAzyme

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