Probing DNA and RNA single molecules with a double optical tweezer

Mangeol, Pierre; Côte, Denis; Bizebard, Thierry; Legrand, Olivier; Bockelmann, Ulrich

doi:10.1140/epje/i2005-10060-4

Cited by 23 publications

(35 citation statements)

References 16 publications

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“…RNA fragments of 0.15-0.2 kb in length carrying the known binding sites of L20C on rRNA and mRNA were designed and linked to two beads via 2.5 kb-long RNA/DNA handles (16) (Fig. 1A, Inset).…”

Section: Resultsmentioning

confidence: 99%

Probing ribosomal protein–RNA interactions with an external force

Mangeol

Bizebard

Chiaruttini

et al. 2011

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

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Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites-i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations -i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by "one-side" clamping. . This approach has been used to probe changes in RNA structure brought about by RNA helicases (12) or the ribosome itself (13), but the effect of proteins that bind specifically and statically to RNA, like r-proteins, has not been studied this way.We focus here on the Escherichia coli r-protein L20 that binds rRNA early during in vitro assembly of the 50S subunit (4). It consists of two domains of similar size-i.e., an N-terminal alphahelical domain that dives deeply into the 50S core and a globular C-terminal domain (L20C) that specifically binds the junction of helices (H) 40-41 (for numbering of rRNA helices and domains see ref. 1) at the exterior of the subunit (2) (Fig. S1). The rplT gene encoding L20 and the upstream rpmI gene encoding L35 are co-transcribed and translationally coupled. By binding to the mRNA at two nearby sites upstream of rpmI, L20 (and L20C) represses the translation of both genes (autogenous control) (14). There are three reasons for choosing L20, and more specifically its globular domain L20C, for this work (see L20 and L20C in SI Text for details). First, whereas many r-proteins (including the full-length L20; Fig. S1) bind different rRNA regions via multiple contacts whose contributions to the overall binding energy are undefined (2, 3), L20C binds a simple rRNA target with nanomolar affinity (14, 15). Second, L20 is dispensable for the function of the ribosome once assembled (4), and therefore the interactions of L20 (and L20C) with rRNA presumably only serves in rRNA folding. Third, the existence of well-characterized binding sites for L20C in both rRNA and mRNA allows comparing the effect of the same protein on the folding of two different RNA molecules.Here, we analyze the effect of L20C on the unfolding of its rRNA and mRNA targets with a mechanical force. The protein dramatically affects the ener...

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Section: Resultsmentioning

confidence: 99%

Probing ribosomal protein–RNA interactions with an external force

Mangeol

Bizebard

Chiaruttini

et al. 2011

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

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“…Different protocols have been reported in the literature to assemble RNA constructs (32,33,38,39,45). These protocols can be divided into several steps that are the focus of this review: (i) Synthesis of the central section of the RNA construct; (ii) synthesis of labeled terminal sections of the RNA construct; (iii) joining of the central and terminal sections of the RNA construct and (iv) isolation and purification of the final RNA construct.…”

Section: Rna Construct Assembly For Single-molecule Force Spectroscopmentioning

confidence: 99%

“…Two main experimental approaches have been adopted for the synthesis of such long central sections; in vitro run-off transcription with RNA polymerase (32,38,39), and polymerization of NDP using Pnpase (33). In vitro run-off transcription has been used in the synthesis of the central sections shorter than 10 kb, and in the synthesis of central sections that contain specific sequences (e.g.…”

Section: The Central Section Synthesis For Ssrna Constructmentioning

confidence: 99%

“…In vitro run-off transcription has been used in the synthesis of the central sections shorter than 10 kb, and in the synthesis of central sections that contain specific sequences (e.g. naturally occurring RNA sequences, RNA structural motifs) (38,39,45). In contrast, Pnpase has been used to synthesize central sections longer than 10 kb, but could be adopted only for random or homopolymeric RNA sequences (33) (Dr R. Tuma, personal communication).…”

Section: The Central Section Synthesis For Ssrna Constructmentioning

confidence: 99%

“…In order to study RNA and RNA-dependent processes with single-molecule force spectroscopic techniques, specialized RNA constructs have to be designed that differ from bulk RNA substrates in several respects: (i) single-molecule force-spectroscopy studies can require long RNAs of lengths greater than ∼1 kb (32,33,38,39,50); (ii) the incorporation of modified nucleotides is often necessary at one or both ends of the RNA construct to immobilize the RNA to a surface or to suspend it between two surfaces, respectively; and (iii) the RNA construct is often composed of different sections that need to be assembled by hybridization or ligation. These specific requirements can present several difficulties for the assembly of an RNA construct.…”

Section: Introductionmentioning

confidence: 99%

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An RNA toolbox for single-molecule force spectroscopy studies

Vilfan

Kamping

Hout

et al. 2007

Nucleic Acids Research

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Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.

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