Correlating Structural Dynamics and Function in Single Ribozyme Molecules

Zhuang, Xiaowei; Kim, Harold; Pereira, Miguel J. B.; Babcock, Hazen P.; Walter, Nils G.; Chu, Steven

doi:10.1126/science.1069013

Cited by 495 publications

(610 citation statements)

References 29 publications

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“…Since then, there has been an explosion of single molecule fluorescence studies on RNA [120]. One of the most puzzling observations in these studies is of molecular "memory" -the fact that different molecules tend to display similar kinetic behavior over long time scales that differ from that of other molecules of the same sequence [81,121]. Future work will undoubtedly address the basis for such quixotic behavior by single molecules.…”

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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“…Different members of a seemingly homogeneous collection of molecules may exhibit different binding kinetics (so-called static disorder), or individual members of a population may exhibit binding kinetics that change over time (dynamic disorder) [11][12][13][14][15][16]. Characterizing these different phenomena and correlating them with information on structural conformation will increase our understanding of the relationship between activity and structure, an important factor in both binding and enzymatic activity [14,[17][18][19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Oijen

2011

Current Opinion in Biotechnology

113

111

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Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van Oijen, A. M. (2011). Single-molecule approaches to characterizing kinetics of biomolecular interactions. Current Opinion in Biotechnology, 22(1), 75-80. DOI: 10.101675-80. DOI: 10. /j.copbio.2010 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Available online at www.sciencedirect.com Single-molecule approaches to characterizing kinetics of biomolecular interactions Antoine M van OijenSingle-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of individual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.

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“…A scanning confocal fluorescence microscope (SCFM) that is sensitive enough to detect single fluorescent molecules can be used to observe single-pair fluorescence resonance energy transfer (spFRET) [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Fluorescence resonance energy transfer (FRET) is a spectroscopic process in which non-radiative energy transfer occurs between an excited dipole (the donor) and another dipole (the acceptor) that has an absorption spectrum that overlaps the emission spectrum of the donor [46].…”

Section: Introductionmentioning

confidence: 99%

“…Changes in the conformation of the molecule are detected as changes in the energy transfer between the donor and acceptor molecule. In this fashion, single molecular-pair FRET has been used to study, e.g., nanometer conformational motions in individual RNA molecules [29][30][31][32][33][34][35], individual DNA molecules [36][37][38][39][40][41] and individual helicases and other motor proteins on DNA [42][43][44][45]. By following changes in FRET efficiency over time, it is possible to observe nanometer distance changes due to macromolecular rearrangements.…”

Section: Introductionmentioning

confidence: 99%

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Zheng¹,

Goldner²,

Leuba³

2007

Methods

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Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/ acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

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Correlating Structural Dynamics and Function in Single Ribozyme Molecules

Cited by 495 publications

References 29 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

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

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