Mg
            <sup>2+</sup>
            -dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

Kim, Harold D.; Nienhaus, G. Ulrich; Ha, Taekjip; Orr, Jeffrey W.; Williamson, James R.; Chu, Steven

doi:10.1073/pnas.032077799

Cited by 255 publications

(284 citation statements)

References 25 publications

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“…Early single molecule FRET studies on RNA folding kinetics revealed a wealth of information inaccessible to traditional pre-steady state kinetic methods, demonstrating the utility of these studies [117][118][119]. Since then, there has been an explosion of single molecule fluorescence studies on RNA [120].…”

Section: Rna: Present and Futurementioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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SummaryThe rapid development of our understanding of the diverse biological roles fulfilled by non-coding RNA has motivated interest in the basic macromolecular behavior, structure, and function of RNA. We focus on two areas in the behavior of complex RNAs. First, we present advances in the understanding of how RNA folding is accomplished in vivo by presenting a mechanism for the action of DEAD-box proteins. Members of this family are intimately associated with almost all cellular processes involving RNA, mediating RNA structural rearrangements and chaperoning their folding. Next, we focus on advances in understanding and characterizing the basic biophysical forces that govern the folding of complex RNAs. Ultimately we expect that a confluence and synergy between these approaches will lead to profound understanding of RNA and its biology.The explosion in our knowledge about noncoding RNA (ncRNA) -RNA that is not translated into protein -has expanded interest in RNA beyond the traditional RNA community. Diverse ncRNAs include the recently-discovered riboswitches, whose novel gene-regulation function is induced by the binding of small metabolites [1]; most of the so-called "Human Accelerated Regions" (HAR) of the human genome, whose recent evolution may be linked to the emergence of the human brain [2]; and many ncRNAs of unknown function [3]. These discoveries underscore the need to understand the fundamental macromolecular behavior of RNA, its structure and dynamics, and how these RNAs are handled and exploited in biology.Study of catalytic RNAs, which allow detection of correct folding via activity measurements, has established basic thermodynamic and kinetic properties of RNA folding. Large catalytic RNAs such as the group I intron from Tetrahymena thermophila and the RNase P RNA from Bacillus subtilis fold at vastly different rates under different conditions upon addition of Mg 2+ and have been shown to fold via multiple parallel pathways, in which different molecules follow different routes with different rates and intermediates, to a common final folded structure [4,5]. These observations support the common view that RNAs traverse rugged energy landscapes as they fold [6,7]. However, little is known about the true shape of these

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Section: Rna: Present and Futurementioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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“…These results, as well as experiments with varying time resolution (8-25 ms, data not shown), indicate that FRET averaging from rapid exchange between states does not occur between the folded and unfolded states so that the observed FRET values reflect those for the unfolded and folded states. 16,17 As K TL represents the formation of the TL/TLR contact without the MC/MCR contact preformed and K′ TL the formation of the contact with the MC/MCR contact preformed (Figure 1c), the ratio of these two equilibrium constants can be used to calculate the tertiary contact cooperativity, ΔG coop , as described in eq 1, which is derived from Figure 1c; that is, how much the formation of one contact favors formation of the other. …”

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confidence: 99%

Direct Measurement of Tertiary Contact Cooperativity in RNA Folding

et al. 2008

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Cooperativity is a central feature of the structure and function of biological macromolecules. It is observed in the folding of proteins to their functional states, in the sharp response of hemoglobin and other proteins to changes in ligand concentration, and in the assembly of macromolecular complexes.Cooperativity in domain formation during protein folding has evolved as a mechanism by which Nature overcomes the difficulty in selectively stabilizing a uniquely folded and functional structure (or small family of structures), among the vast number of partially folded and nonfunctional states that would likely dominate if each of the weak long-range domain contacts were to form independently. This thermodynamic scenario is well-documented for single domain proteins (e.g., refs 1-3 ) and is typically measured using double mutant cycles, analogous to the mutant cycle shown in Figure 1c.Despite its fundamental importance, cooperativity is not always employed in all aspects of protein folding. Regions of proteins often form and break up as units, and these units are sometimes referred to as domains. Further, the cooperativity between domains can vary, from high, for a protein with extensive interfaces and reinforcing interactions, 4 to nonexistent, for a protein like titin with individual domains that are noninteracting "beads on a string". 5 RNA, like proteins, must often fold to distinct three-dimensional structures to carry out biological functions. However, RNA forms stable secondary structure in the absence of tertiary structure, indicating some limits to cooperativity in RNA tertiary folding. Indeed, one might consider folding of an RNA from a preformed secondary structure to a functional tertiary structure as akin to folding of a multidomain protein. The fundamental question then arises: To what extent is there cooperativity in RNA folding?We determined the tertiary contact cooperativity for the independently folding P4-P6 domain derived from the T. thermophila group I intron 6 (Figure 1a,b) using a single molecule fluorescence energy transfer (smFRET) folding assay. The P4-P6 crystal structure in 1996 revealed, for the first time, the side-by-side packing of RNA helices. 7 These helices are connected by a junction (J5/5a) and joined by two regions of tertiary contact, the metal core/ metal core receptor (MC/MCR, Figure 1a, Figure 1a,b, magenta). [6][7][8][9][10] We refer to these regions as "tertiary contacts", and we quantitatively investigate the energetic crosstalk between these tertiary contacts.A thermodynamic scheme for determining the tertiary contact cooperativity in P4-P6 is shown in Figure 1c. The unfolded ensemble (U) comprises the large number of conformations with secondary structure but no tertiary contacts. The unfolded ensemble is in equilibrium with the fully folded state , which has both tertiary contacts formed, and this equilibrium is described by K fold . The two intermediate species, I TL and I MC , have only one tertiary contact formed. In I TL , the tetraloop/receptor contact is formed...

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“…[3][4][5][6] In particular, intramolecular distances can now be probed by attaching dye molecules to sites of interest and measuring the efficiency of fluorescence resonance energy transfer (FRET). 3,5 This approach has been applied to monitor folding of biopolymers (proteins and nucleic acids), [7][8][9] enzyme-substrate complex formation, [10][11][12][13][14] and the operation of molecular motors. [15][16][17][18][19][20] A persistent issue in such studies is that they explicitly follow the dynamics of one, or at most a few, of the many degrees of freedom of a macromolecular system.…”

Section: Introductionmentioning

confidence: 99%

Models of Single-Molecule Experiments with Periodic Perturbations Reveal Hidden Dynamics in RNA Folding

Liu

et al. 2009

J. Phys. Chem. B

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Traditionally, microscopic fluctuations of molecules have been probed by measuring responses of an ensemble to perturbations. Now, single-molecule experiments are capable of following fluctuations without introducing perturbations. However, dynamics not readily sampled at equilibrium should be accessible to nonequilibrium single-molecule measurements. In a recent study [Qu, X. et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6602-6607], the efficiency of fluorescence resonance energy transfer (FRET) between probes on the L18 loop and 3′ terminus of the 260 nucleotide RNase P RNA from Bacillus stearothermophilus was found to exhibit complex kinetics that depended on the (periodically alternating) concentration of magnesium ions ([Mg 2+ ]) in solution. Specifically, this time series was found to exhibit a quasi-periodic response to a squarewave pattern of [Mg 2+ ] changes. Because these experiments directly probe only one of the many degrees of freedom in the macromolecule, models are needed to interpret these data. We find that Hidden Markov Models are inadequate for describing the nonequilibrium dynamics, but they serve as starting points for the construction of models in which a discrete observable degree of freedom is coupled to a continuously evolving (hidden) variable. Consideration of several models of this general form indicates that the quasi-periodic response in the nonequilibrium experiments results from the switching (back and forth) in positions of the minima of the effective potential for the hidden variable. This switching drives oscillation of that variable and synchronizes the population to the changing [Mg 2+ ]. We set the models in the context of earlier theoretical and experimental studies and conclude that single-molecule experiments with periodic peturbations can indeed yield qualitatively new information beyond that obtained at equilibrium.

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Mg ²⁺ -dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

Cited by 255 publications

References 25 publications

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Direct Measurement of Tertiary Contact Cooperativity in RNA Folding

Models of Single-Molecule Experiments with Periodic Perturbations Reveal Hidden Dynamics in RNA Folding

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Mg 2+ -dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

Cited by 255 publications

References 25 publications

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Direct Measurement of Tertiary Contact Cooperativity in RNA Folding

Models of Single-Molecule Experiments with Periodic Perturbations Reveal Hidden Dynamics in RNA Folding

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Mg ²⁺ -dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules