Ion-induced folding of the hammerhead ribozyme: afluorescence resonance energy transfer study

Bassi, Gurminder S.; Murchie, Alastair I. H.; Walter, Frank; Clegg, Robert M.; Lilley, David M. J.

doi:10.1093/emboj/16.24.7481

Cited by 155 publications

(168 citation statements)

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“…In previous measurements with HHR at pH 6, an even higher apparent Mg 2+ dissociation constant was found than in our measurements at pH 8, indicating a possible pH dependence of Mg 2+ binding (24). Steady-state fluorescence spectroscopy has been previously used to determine the mean FRET efficiency of fluorophorelabeled hammerhead ribozymes (7,8). These steady-state experiments, however, can only provide qualitative information about the fluorophore distances, whereas a technique such as time-resolved FRET (tr-FRET) yields quantitative distances between the donor and acceptor fluorophores (28,30).…”

Section: Resultsmentioning

confidence: 99%

“…Earlier solution studies of the hammerhead ribozyme have used steady-state FRET to measure the efficiency of a single energy transfer between two fluorophores attached to stems I and II (7,8,29). This energy transfer efficiency is related to the mean stem I-stem II distance derived from our tr-FRET measurements.…”

Section: Discussionmentioning

confidence: 98%

“…To investigate the roles of metal ions, conformational changes, and this A‚U tandem in the folding and function of the hammerhead ribozyme, we have carried out a comparative study between two constructs, HHL and HHR. Ribozyme HHL resembles a three-strand construct used by Lilley and co-workers to study the Mg 2+ dependence of folding by steady-state FRET (8). Ribozyme HHR is similar to the higher-activity ribozyme described by Clouet-d'Orval and Uhlenbeck (24,25).…”

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confidence: 95%

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Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

et al. 2003

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The hammerhead ribozyme is one of the best-studied small RNA enzymes, yet is mechanistically still poorly understood. We measured the Mg 2+ dependencies of folding and catalysis for two distinct hammerhead ribozymes, HHL and HHR. HHL has three long helical stems and was previously used to characterize Mg 2+ -induced folding. HHR has shorter stems and an A‚U tandem next to the cleavage site that increases activity ∼10-fold at 10 mM Mg 2+ . We find that both ribozymes cleave with fast rates (5-10 min -1 , at pH 8 and 25°C) at nonphysiologically high Mg 2+ concentrations, but with distinct Mg 2+ dissociation constants for catalysis: 90 mM for HHL and 10 mM for HHR. Using time-resolved fluorescence resonance energy transfer, we measured the stem I-stem II distance distribution as a function of Mg 2+ concentration, in the presence and absence of 100 mM Na + , at 4 and 25°C. Our data show two structural transitions. The larger transition (with Mg 2+ dissociation constants in the physiological range of ∼1 mM, below the catalytic dissociation constants) brings stems I and II close together and is hindered by Na + . The second, globally minor, rearrangement coincides with catalytic activation and is not hindered by Na + . Additionally, the more active HHR exhibits a higher flexibility than HHL under all conditions. Finally, both ribozyme-product complexes have a bimodal stem I-stem II distance distribution, suggesting a fast equilibrium between distinct conformers. We propose that the role of diffusely bound Mg 2+ is to increase the probability of formation of a properly aligned catalytic core, thus compensating for the absence of naturally occurring kissing-loop interactions.Hammerhead ribozymes are found in a number of plant pathogenic viroids and virusoids, where they are involved in self-cleavage and self-ligation of the plus and minus strands during the pathogen's genome replication (1, 2). In addition, the hammerhead ribozyme has been found to mediate self-cleavage of satellite DNA transcripts in the newt (3). Several hammerhead crystal structures have been determined (4-6), and the global structure was confirmed in solution by steady-state fluorescence resonance energy transfer (FRET) 1 (7,8), gel electrophoresis (9), and transient electric birefringence (10) (Figure 1). The ribozyme consists of three helical stems arranged in a "Y-shape", with 11 nucleotides at the junction forming the highly conserved catalytic core (11,12) (Figure 1). The catalytic core is comprised of two domains; stems I and II are arranged in an acute angle by a sharp uridine turn in the backbone trajectory of domain 1, while stems II and III stack coaxially with the formation of two G‚A and one A‚U non-WatsonCrick base pairs in domain 2. The cleavage site is located 3′ to C17, which is buttressed to domain 1 by a single hydrogen bond with C3 (Figure 1). † This work was supported by NIH Grant GM62357-01 to N.G.W., a postdoctoral fellowship from the Swiss National Funds to D.R., a DAAD scholarship to K.W., and a University of Michig...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 98%

mentioning

confidence: 95%

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Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

et al. 2003

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“…Fully deprotected oligonucleotides for fluorescence spectroscopy were purified by gel electrophoresis as described previously (12). Fluorescein (PE-ABI) and Cy3 (Glen Research) were coupled to the 5′-termini as phosphoramid- † …”

Section: Construction Of Dna Duplexes For Fluorescence and Nmr Studiesmentioning

confidence: 99%

“…Fluorescent spectra were obtained at 4°C using an SLM 8100 fluorimeter (SLM-Aminco, Urbana, IL) as described previously (12). Values of E FRET were measured using the acceptor normalization method (1,21).…”

Section: Fluorimetry and Measurements Of Fluorescence Resonance Energmentioning

confidence: 99%

Location of Cyanine-3 on Double-Stranded DNA: Importance for Fluorescence Resonance Energy Transfer Studies

et al. 2000

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Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Förster length for this pair on DNA is 56 Å, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5′-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.Fluorescence resonance energy transfer (FRET) has been applied to the analysis of global conformation and folding transitions in nucleic acids with increasing frequency in the past decade. Potentially, the method provides distance information between known points that is typically in the range of 10-80 Å. This is a range that is inaccessible to other solution methods, and thus, the approach is complementary in principle to NMR in particular. FRET has been applied to the analysis of DNA structures (1-10) and more recently to the folding of a number of interesting RNA structures (11-18).Determination of either relative or absolute distance information using FRET depends on the distance-dependent transfer of excitation energy from a donor to an acceptor fluorophore, resulting from the coupling between their transition dipoles. The efficiency of the transfer (E FRET ) depends inversely on the sixth power of the distance separating the fluorophores (19), and is given by where R is the scalar distance between the fluorophores and R 0 is the characteristic Förster distance for the donoracceptor combination at which E FRET equals 0.5. In general in our studies, we attach the fluorophores to the 5′-termini of known helical arms with a constant terminal sequence, and in this way, we can compare different end-to-end distances and study how these change upon the induction of conformational change.Most FRET studies of nucleic acids have used fluorescein as the donor, and many have used tetramethylrhodamine as the acceptor. Fluorescein is generally very mobile when attached to DNA or RNA, and thus, orientation is not a problem in the interpretation of the measured FRET efficiencies. However, tetramethylrhodamine has some disadvantages, including the difficulty of coupling the fluorophore to the nucleic acid as a phosphoramidite. For this reason, we have largely changed to the use of indocarbocyanine-3 (Cy3) as the acceptor in our recent studies (F...

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Modified oligoribonucleotides as site-specific probes of RNA structure and function

Earnshaw

Gait

1998

Biopolymers

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Ion-induced folding of the hammerhead ribozyme: afluorescence resonance energy transfer study

Cited by 155 publications

References 52 publications

Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Location of Cyanine-3 on Double-Stranded DNA: Importance for Fluorescence Resonance Energy Transfer Studies

Modified oligoribonucleotides as site-specific probes of RNA structure and function

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Ion-induced folding of the hammerhead ribozyme: afluorescence resonance energy transfer study

Cited by 155 publications

References 52 publications

Diffusely Bound Mg2+ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Diffusely Bound Mg2+ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Location of Cyanine-3 on Double-Stranded DNA: Importance for Fluorescence Resonance Energy Transfer Studies

Modified oligoribonucleotides as site-specific probes of RNA structure and function

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Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core