Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA

Abels, J.; Moreno‐Herrero, Fernando; Heijden, Thijn van der; Dekker, Cees; Dekker, Nynke H.

doi:10.1529/biophysj.104.052811

Cited by 241 publications

(250 citation statements)

References 31 publications

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“…From fits of the WLC model, we determined the contour length L C = 1.15 ± 0.02 μm and the bending persistence length A RNA = 57 ± 2 nm in the presence of 100 mM monovalent salt (SI Appendix, Fig. S2A), in good agreement with the expected length (1.16 μm, assuming 0.28 nm per bp) (22,23) and previous single-molecule stretching experiments (15,16). A RNA decreases with increasing ionic strength (16) (SI Appendix, Fig.…”

Section: Resultssupporting

confidence: 81%

“…1A). Although recent single-molecule stretching experiments using torsionally unconstrained dsRNA have revealed its bending persistence length (15,16), stretch modulus (16), and an overstretching transition (16,17), its response to torsional strains and structural transitions under forces and torques is unknown. This dearth of information on dsRNA is partially due to the relative difficulty, compared with dsDNA, of assembling RNA constructs suitable for single-molecule force and torque measurements.…”

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confidence: 99%

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Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Lipfert

Skinner

Keegstra

et al. 2014

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

170

258

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RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a forcetorque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed "P-RNA," and a highly underwound, left-handed state denoted "L-RNA." Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA-protein interactions at the single-molecule level.RNA | nucleic acids | magnetic tweezers | force | torque

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Section: Resultssupporting

confidence: 81%

mentioning

confidence: 99%

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Lipfert

Skinner

Keegstra

et al. 2014

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

170

258

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“…As shown in the model of RNA exit (Fig. 4), RNA bent toward the dock domain-contacting position requires larger angles than that bent toward Rpb7 and thus is energetically unfavorable because the physics of bending RNA demands energy of k B T(⌬ ) 2 ( /2L), where is the persistent length of ssRNA, Ϸ1-1.4 nm, and L is the contour length of the ssRNA (39,40). Of course, interactions of RNA-Rpb7 and those between RNA and dock domain must be taken into account to complete the analysis.…”

Section: Discussionmentioning

confidence: 99%

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Chen¹,

Chang²,

Yen³

et al. 2009

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

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A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5 end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5 end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.nanometry ͉ structure ͉ transcription ͉ in-gel ͉ single-molecule fluorescence R NA polymerase II (pol II), a protein complex containing 12 subunits, Rpb1-Rpb12, of a total mass of Ϸ500 kDa and size Ϸ100-140 Å, is the enzyme machinery synthesizing mRNA in all eukaryotes (1). X-ray studies of pol II complexes (2-4) led to an atomic model containing structural elements with functional implications (Fig. 1A). In a transcribing pol II, between the ''clamp'' and ''jaw'' domain, lies a cleft (4) that harbors the active center, a straight duplex DNA and an RNA-DNA hybrid (position ϩ1 to Ϫ8, ϩ1 denoting the nucleotide addition site). The strand separation of RNA from DNA template occurs upstream of the hybrid at positions Ϫ9 and Ϫ10, facilitated by a set of protein loops including the ''lid'' domain as a driving wedge. Nascent RNA moves through an exit pore from the active center, crossing a saddle-like surface, beneath an ''arch'' bridging the clamp and wall (5).How does pol II instruct the nascent RNA to exit beyond the saddle? Is there a unique path on pol II connecting the active center to its exterior that nascent RNA may follow? To date, insights into the RNA exit have come from analysis of pol II surface charge distribution: two positively charged grooves, on either side of the ''dock domain'' (Fig. 1 A), can accommodate ssRNA (5). One groove, putatively referred as ''exit channel 1,'' runs around the base of the clamp, leading toward the stalk of subcomplex Rpb4-Rpb7, which can bind RNA via its ribonucleoprotein fold (6, 7). The other groove, termed ''exit channel 2,'' runs down the back side of pol II, through Rpb3 and Rpb11, leading toward Rpb8, a subunit equally competent in RNA binding by its single-strand nucleic acid-binding motif. Intriguingly, exit channel 1 would cause the RNA to bend sharply, implying that channel 2 is energetically favored for RNA binding. Yet, evidence in support of the channel 1 hypothesis has come from observation...

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“…(Fig. 1b) have been used to study the mechanical properties of nucleic acids (Strick et al 1996;Abels et al 2005) and the mechanisms of enzymatic reactions with DNAs as substrates (Crisona et al 2000;Revyakin et al 2003;Dessinges et al 2004;Gore et al 2006). In a recent study, Abels et al (2005) ligated a long dsRNA (4·2 kb or 8·3 kb) with two 0·4 kb pieces of dsRNA, each of which was labeled by multiple digoxigenins or biotins.…”

Section: Instrumentationmentioning

confidence: 99%

Determination of thermodynamics and kinetics of RNA reactions by force

Tinoco

Bustamante

2006

Quart. Rev. Biophys.

107

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Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.

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Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA

Cited by 241 publications

References 31 publications

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Determination of thermodynamics and kinetics of RNA reactions by force

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