The Structural Basis of Hammerhead Ribozyme Self-Cleavage

Murray, James B.; Terwey, Daniel P; Maloney, Lara; Karpeisky, Alexander; Usman, Nassim; Beigelman, Leonid; Scott, William G.

doi:10.1016/s0092-8674(00)81134-4

Cited by 228 publications

(240 citation statements)

References 44 publications

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“…The second, globally minor, structural adjustment coincides with activation of the ribozyme at high Mg 2+ concentrations (90 mM for HHL and 10 mM for HHR). Our data are therefore largely consistent with the notions, suggested first by X-ray crystallography, that the functionally relevant conformation has stems I and II at an acute angle and that only a relatively small conformational change is required to reach the catalytic transition state (5,17,22,43). Additionally, we note that throughout the entire Mg 2+ range, HHR has a more flexible stem I-stem II distance than HHL (Figure 6), an observation that may relate to its higher catalytic activity, consistent with the activation entropy increase previously reported (25).…”

Section: Discussionsupporting

confidence: 91%

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: Discussionsupporting

confidence: 91%

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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“…5 were achieved using a near-axial ZFS tensor (E/D = 0.1), a finding supported by the axial geometry observed in the crystal structure of the Mn(II)EDTA complex [112]. As this species serves to calibrate our water-counting measurements via ESEEM spectroscopy, the MF EPR spectroscopic characterization of Mn(II)EDTA presented here is essential to extending these types of studies to higher frequencies [98,114].In conclusion, the MF EPR approach is not only useful in determining spin Hamiltonian parameters for paramagnetic systems (Eq. (2)) but may also provide clues as the degree of homogeneity associated with the binding site of the metal cofactor.…”

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confidence: 60%

“…Biochemical and EPR spectroscopic studies carried out by DeRose and coworkers [92,94] found that only one of these sites tightly binds its respective metal ion (k d ≤ 10 µM), and that this site must be populated for "catalytic" activity. However, all available spectroscopic evidence indirectly suggested that the high-affinity binding site exists between the N7 of a guanine residue (G10.1) and the phosphate oxygen of an adjacent adenine (A9), about 2.0 nm from the site of RNA cleavage [35,92,[95][96][97][98].Only recently has this tentative assignment been confirmed through ESEEM spectroscopic measurements of site-specific isotope labeling of the G10.1 nitrogens [99]. Herein, we report results from EPR spectroscopic experiments at multiple frequencies to study a set of simple nucleotide and RNA Mn(II) complexes.…”

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confidence: 99%

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

et al. 2007

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Electron paramagnetic resonance studies at multiple frequencies (MF EPR) can provide detailed electronic structure descriptions of unpaired electrons in organic radicals, inorganic complexes, and metalloenzymes. Analysis of these properties aids in the assignment of the chemical environment surrounding the paramagnet and provides mechanistic insight into the chemical reactions in which these systems take part. Herein, we present results from pulsed EPR studies performed at three different frequencies (9, 31, and 130 GHz) on [Mn(II)(H 2 O) 6 ] 2+ , Mn(II) adducts with the nucleotides ATP and GMP, and the Mn(II)-bound form of the hammerhead ribozyme (MnHH). Through line shape analysis and interpretation of the zero-field splitting values derived from successful simulations of the corresponding continuous-wave and field-swept echodetected spectra, these data are used to exemplify the ability of the MF EPR approach in distinguishing the nature of the first ligand sphere. A survey of recent results from pulsed EPR, as well as pulsed electron-nuclear double resonance and electron spin echo envelope modulation spectroscopic studies applied to Mn(II)-dependent systems, is also presented. Mn-Containing Biological SystemsManganese operates as a cofactor in numerous proteins, serving both catalytic and structural roles [1][2][3][4]. Many Mn-dependent enzymes take advantage o f the rich redox chemistry available to the metal, accessing the +2, +3, +4, and perhaps even the +5 oxidation states during their turnover. For example, Mn-superoxide dismutase (MnSOD), which detoxifies the cell of the superoxide radical , cycles between the Mn(II) and Mn(III) oxidation states via the ping-pong type mechanism shown below [5][6][7][8][9].(1a) (1b) Other examples of such mononuclear redox-active enzymes include the manganese peroxidase responsible for lignin degradation by white-rot fungus [10][11][12]; a unique Mndependent form of lipoxygenase [13][14][15][16]; oxalate decarboxylase [17,18]; as well as an extradiol catechol dioxygenase [19][20][21]. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn order to help minimize kinetic and thermodynamic penalties associated with electron transfer events and the execution of chemical reactions, two or more metal centers can be coupled together to provide active sites capable of conducting multiple electrons while still operating within a physiologically accessible range of reduction potentials [22]. Only three such examples of redox-active polynuclear Mn enzymes are known: Mn-catalase that disproportionates hydrogen peroxide [23][24][25][26]; a Mn form of ribonucleotide reductase (Mn-RNR) [27,28]; and, arguably the most recognized Mn-dependent enzyme, the photosynthetic core of green plants, algae, and certain cyanobacteria termed photosystem II (PSII) [29,30]. Through a series of photoinitiated electron transfer events, oxidizing equivalents are stored on the tetranuclear Mn core of PSII -the oxygen-evolving complex (OEC) -which then extracts four electr...

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“…The final ribose MePA 2− residue was fit within the RNA sequence UCA taken from the near in-line geometry in the x-ray crystal structure of a hammerhead ribozyme "late-intermediate". 74 ABNR minimization was performed for this sequence with the U and A residues fixed and where necessary, small adjustments to the internal parameters were made to improve fitting. Because the pentacoordinated geometry of the phosphorane is quite rigid, the dihedrals had negligible effect, and hence were set to zero.…”

Section: Phosphate Transition State Analogmentioning

confidence: 99%

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

et al. 2006

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Force field parameters specifically optimized for residues important in the study of RNA catalysis are derived from density-functional calculations in a fashion consistent with the CHARMM27 allatom empirical force field. Parameters are presented for residues that model reactive RNA intermediates and transition state analogs, thio-substituted phosphates and phosphoranes, and bound Mg 2+ and di-metal bridge complexes. Target data was generated via density-functional calculations at the B3LYP/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) level. Partial atomic charges were initially derived from the CHelpG electrostatic potential fitting and subsequently adjusted to be consistent with the CHARMM27 charges and Lennard-Jones parameters were determined to reproduce interaction energies with water molecules. Bond, angle and torsion parameters were derived from the density-functional calculations and renormalized to maintain compatibility with the existing CHARMM27 parameters for standard residues. The extension of the CHARMM27 force field parameters for the non-standard biological residues presented here will have considerable use in simulations of ribozymes, including the study of freeze-trapped catalytic intermediates, metal ion binding and occupation, and thio effects.

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The Structural Basis of Hammerhead Ribozyme Self-Cleavage

Cited by 228 publications

References 44 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

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

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The Structural Basis of Hammerhead Ribozyme Self-Cleavage

Cited by 228 publications

References 44 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

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

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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