Adjacent Phosphate Groups Research Articles

Theoretical investigation of the effect of phosphate doping on the aggregation of Au atoms on an Al2O3 (0001) surface

Using density functional theory, the adsorption of Au atoms and Au dimers onto α-Al2O3 (0001) surfaces with various phosphate coverages was investigated. When the phosphate coverage is low (0% or 25%), Au dimers are more stable than isolated Au atoms on the surfaces, and therefore, aggregation of Au atoms into Au dimers is exothermic. In contrast, when the phosphate coverage is high (50% or 100%), isolated Au atoms are more stable than Au dimers on the surface, and Au atom aggregation is endothermic. This difference is caused by differences in the distance between adjacent phosphate groups; at certain distances, the phosphate groups are capable of bridging Au atoms. The Au-Au bond of a Au dimer at a phosphate-phosphate bridge site is destabilized by hole transfer from the Al2O3 surfaces to the bonding σ orbital of the Au dimer. Isolated phosphate groups cannot cause the destabilization. These results provide a good explanation for the mechanism of the suppression of aggregation of Au particles caused by phosphate doping on an Al2O3 surface, and show the importance of charge transfer from a surface to Au atoms for suppressing Au sintering.

Sequestering Ability of Oligophosphate Ligands toward Al3+ in Aqueous Solution

The interactions between Al3+ and ortho-phosphate (PO43–), pyro-phosphate (P2O74–, PP), tripolyphosphate (P3O105–, TPP) and hexa-metaphosphate (P6O186–, HMP) in aqueous solution have been studied. Formation constants, speciation models, and possible structures for the complexes formed are discussed on the basis of potentiometric, calorimetric, 31P–{1H} NMR, laser desorption mass spectrometric (LD MS) and MS/MS results. 31P–{1H} NMR experiments for Al3+–TPP system are in agreement with the speciation model drawn from analysis of potentiometric titration data, suggesting that TPP binds to Al3+ with two adjacent phosphate oxygen atoms, forming a six-membered ring. LD MS has provided identification and structural information for Al3+–PP, −TPP, and −HMP complexes which supports the reliability of speciation models proposed on the basis of the potentiometric data. Moreover, MS/MS spectra obtained from [ML]+ precursor ion show that the ion species involve linear HMP isomers and that two adjacent terminal phosphate groups probably bind Al3+. Standard enthalpy change values were obtained by titration calorimetry; all are endothermic, as is typical for hard–hard interactions. The dependence of formation constants on ionic strength over the range I = 0.1–1 mol kg–1 is also reported. The sequestering ability of all the ligands under study toward Al3+ was also evaluated by the empirical parameter pL0.5.

Increased intracellular Ca2+ concentrations prevent membrane localization of PH domains through the formation of Ca2+-phosphoinositides

Insulin resistance, a key etiological factor in metabolic syndrome, is closely linked to ectopic lipid accumulation and increased intracellular Ca2+ concentrations in muscle and liver. However, the mechanism by which dysregulated intracellular Ca2+ homeostasis causes insulin resistance remains elusive. Here, we show that increased intracellular Ca2+ acts as a negative regulator of insulin signaling. Chronic intracellular Ca2+ overload in hepatocytes during obesity and hyperlipidemia attenuates the phosphorylation of protein kinase B (Akt) and its key downstream signaling molecules by inhibiting membrane localization of pleckstrin homology (PH) domains. Pharmacological approaches showed that elevated intracellular Ca2+ inhibits insulin-stimulated Akt phosphorylation and abrogates membrane localization of various PH domain proteins such as phospholipase Cδ and insulin receptor substrate 1, suggesting a common mechanism inhibiting the membrane targeting of PH domains. PH domain-lipid overlay assays confirmed that Ca2+ abolishes the binding of various PH domains to phosphoinositides (PIPs) with two adjacent phosphate groups, such as PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 Finally, thermodynamic analysis of the binding interaction showed that Ca2+-mediated inhibition of targeting PH domains to the membrane resulted from the tight binding of Ca2+ rather than PH domains to PIPs forming Ca2+-PIPs. Thus, Ca2+-PIPs prevent the recognition of PIPs by PH domains, potentially due to electrostatic repulsion between positively charged side chains in PH domains and the Ca2+-PIPs. Our findings provide a mechanistic link between intracellular Ca2+ dysregulation and Akt inactivation in insulin resistance.

Insights into the activation mechanism of class I HDAC complexes by inositol phosphates.

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.

Replacement of water molecules in a phosphate binding site by furanoside-appended lin-benzoguanine ligands of tRNA-guanine transglycosylase (TGT).

The enzyme tRNA-guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure-based design for this enzyme is the filling of the polar ribose-34 pocket. Herein, we describe a novel series of ligands consisting of furanoside-appended lin-benzoguanines. They were designed to replace a conserved water cluster and differ by the functional groups at C(2) and C(3) of the furanosyl moiety being either OH or OMe. The unfavorable desolvation of Asp102 and Asp280, which are located close to the ribose-34 pocket, had a significant impact on binding affinity. While the enzyme has tRNA as its natural substrate, X-ray co-crystal structures revealed that the furanosyl moieties of the ligands are not accommodated in the tRNA ribose-34 site, but at the location of the adjacent phosphate group. A remarkable similarity of the position of the oxygen atoms in these two structures suggests furanosides as a potential phosphate isoster.

Photochemical Modulation of DNA Conformation by Organic Dications

A group of azobenzene derivatives containing two quaternary ammonium groups with various intercharge distances between them was synthesised and used to control photochemically the conformation of genomic DNA by switching the distance between cationic ammonium groups in the dications. It was found that isomerisation of either dication from the trans form to cis resulted in an increase in the dication's efficiency for DNA compaction; this is associated with a decrease in intercharge distance between ammonium groups and leads to a better match of the binder's cationic groups to adjacent phosphate groups of DNA. Ammonium dications have several important advantages over the photosensitive surfactant type of diazobenzene reported earlier: they can be used at significantly lower (>100-fold) concentrations than photosensitive surfactants, and DNA conformation control can be performed over a broader concentration range of dications. The influence of intercharge distance in photosensitive dications on photo-induced DNA binding discrimination is discussed, and the molecular mechanism is proposed.

Phosphorus-containing liquid cycloaliphatic epoxy resins for reworkable environment-friendly electronic packaging materials

Novel thermally reworkable, phosphorus-containing, di- and tri-functional liquid cycloaliphatic epoxy resins were designed and synthesized. Their chemical structures were characterized by means of MS, FTIR, 1H NMR and 31P NMR methods. After curing, the products were transparent and stable up to 220 °C, while exhibited quick thermal decomposition at the temperature range of 255–280 °C. The removal test showed that, after heat-treatment at 260 °C in air atmosphere for only 4 min, the residual char on the glass substrate could be easily wiped off. This unique degradation behavior was attributed to the synergistic effect of two factors: the thermally-labile phosphate groups evenly distributed within the three-dimensional network and the in-situ catalyzing of phosphoric acid generated from the cleavage of phosphate bond on the pyrolysis of adjacent other phosphate and ester groups, as evidenced by the results of molecular modeling, isothermal TGA and FTIR spectra. In addition, compared to the commercial cycloaliphatic epoxy resin ERL-4221, the newly synthesized epoxy resins had increased limiting oxygen index (LOI) by 31%. The combination of excellent reworkability, non-halogen flame retardancy, high glass transition temperature of 227 °C and high mechanical modulus endows them the potential for environment-friendly microelectronic and optoelectronic packaging applications.

Hydration Effects on the Microwave Dielectricity in Dry Poly(dA)–Poly(dT) DNA

Using a microwave cavity-perturbation method, the temperature dependence of the complex dielectric constant (\(\tilde{\epsilon}=\epsilon_{1}+\mathrm{i}\epsilon_{2}\)) at 16.3 GHz is measured on a dry poly(dA)–poly(dT) DNA sample prepared under three-different relative humidity (RH) of 0, 11 and 93%. For RH=0%, the temperature dependence of both e 1 and e 2 are explained by the rotational motion of water molecules electrostatically bonding to the negatively charged phosphate group in the primary hydration shell. The activation-type behavior in e 2 is discussed with a double-well potential for a process that the electric dipole of the hydrated water is thermally rotated to the adjacent phosphate groups. For RH=11%, e 2 takes a broad maximum at 280 K due to the collective motion of water molecules in the primary hydration shell. While these features qualitatively resemble to those of free water, the absolute values are much small due to an extremely small number of hydrated water molecules. Moreover the conf...

Ca2+-Inositol Phosphate Chelation Mediates the Substrate Specificity of β-Propeller Phytase

Inositol phosphates are recognized as having diverse and critical roles in biological systems. In this report, kinetic studies and TLC analysis indicate that beta-propeller phytase is a special class of inositol phosphatase that preferentially recognizes a bidentate (P-Ca(2+)-P) formed between Ca(2+) and two adjacent phosphate groups of its natural substrate phytate (InsP(6)). The specific recognition of a bidentate chelation enables the enzyme to sequentially hydrolyze one of the phosphate groups in a bidentate of Ca(2+)-InsP(6) to yield a myo-inositol trisphosphate (InsP(3)) and three phosphates as the final products. A comparative analysis of (1)H- and (13)C NMR spectroscopy with the aid of 2D NMR confirms that the chemical structure of the final product is myo-Ins(2,4,6)P(3). The catalytic properties of the enzyme suggest a potential model for how the enzyme specifically recognizes its substrate Ca(2+)-InsP(6) and produces myo-Ins(2,4,6)P(3) from Ca(2+)-InsP(6). These findings potentially provide evidence for a selective Ca(2+)-InsPs chelation between Ca(2+) and two adjacent phosphate groups of inositol phosphates.

Oligomeric Surfactant and Triblock Copolymer Syntheses of Aluminum Organophosphonates with Ordered Mesoporous Structures

Mesostructural control of aluminum organophosphonates (AOPs) with methylene groups inside the inorganic−organic hybrid frameworks was performed by using oligomeric surfactants (C16EO10 and C16EO20) and triblock copolymers (EO80PO30EO80, EO106PO70EO106, and EO20PO70EO20). The templating by oligomeric surfactants with hexadecyl chains that directed the formation of 2-D hexagonal mesostructured and mesoporous AOPs (BET surface areas; ∼579 m2 g-1, pore volumes; ∼0.53 cm3 g-1), especially highly ordered materials, can be obtained by using C16EO20. Mesoporous materials with 2-D hexagonal phases (∼279 m2 g-1, ∼0.76 cm3 g-1) were also prepared through triblock copolymer templating. The pore diameters (6.0−9.2 nm) were largely expanded by using triblock copolymers as compared with those of oligomeric surfactant templated materials (3.3−3.8 nm). Low-temperature calcination of the mesostructured AOPs was useful for removing surfactant molecules to afford periodic non-silica-based hybrid mesoporous materials. Once internal organic moieties are eliminated by calcination at high temperatures, the frameworks are composed of only inorganic units containing two adjacent phosphate groups (−P−OH + HO−P−). Further condensation in the inorganic frameworks is therefore not preferable. Inorganic units are supposed to be restructured to form aluminophosphate-like frameworks (−Al−O−P−) composed of alternating AlO4 and PO4 units and then the mesostructures collapse. Therefore, mesostructural stabilities of AOPs are related to stabilities of methylenediphosphonate groups (⋮PCH2P⋮) and the inner organic groups of the hybrid frameworks.

Non-cognate Enzyme–DNA Complex: Structural and Kinetic Analysis of EcoRV Endonuclease Bound to the EcoRI Recognition Site GAATTC

The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.

Interaction between ferric ions, phospholipid hydroperoxides, and the lipid phosphate moiety at physiological pH

Iron-catalyzed lipid peroxidation was examined using 1H NMR in a biphasic aqueous-chloroform system. At physiological pH (7.4), mole ratios of phospholipids/Fe3+ as low as 1300:1 catalyzed the rapid disappearance of endogenous lipid hydroperoxides with a loss of two of the four double bonds in PC containing palmitic (16:0) and arachidonic (20:4) acids in the sn-1 and sn-2 positions, respectively. The predominant phospholipid products after 1 h at 20 degrees C were a 9-carbon mono-unsaturated carbonyl and a phospholipid with an 11-carbon delta5,8 FA in the sn-2 position. PC with linoleic acid (18:2) in the sn-2 position lost one double bond and formed a phospholipid with a 9-carbon FA. Cardiolipin (linoleic acid-rich) also lost about 40% of its double bonds. No detectable loss was seen for PC containing oleic acid (18:1) or neutral lipids with PUFA. At arachidonyl PC/Fe3+ ratios less than 20:1, significant broadening of the choline methyl proton peak was evident, indicating that Fe3+ may form a complex with the adjacent phosphate group and that the complex involves both the phosphate and the hydroperoxide adjacent to the delta11 double bond. The results demonstrate that, at physiological pH, Fe3+-catalyzed peroxidation in polyunsaturated phospholipids occurs selectively adjacent to specific double bonds (delta9 or delta11). These PC-derived products have been shown to activate components of the inflammatory system. This suggests that the episodic release of ferric ions may play a significant role in generating inflammatory mediators.

Folding of the Tetrahymena ribozyme by polyamines: importance of counterion valence and size.

Polyamines are abundant metabolites that directly influence gene expression. Although the role of polyamines in DNA condensation is well known, their role in RNA folding is less understood. Non-denaturing gel electrophoresis was used to monitor the equilibrium folding transitions of the Tetrahymena ribozyme in the presence of polyamines. All of the polyamines tested induce near-native structures that readily convert to the native conformation in Mg(2+). The stability of the folded structure increases with the charge of the polyamine and decreases with the size of the polyamine. When the counterion excluded volume becomes large, the transition to the native state does not go to completion even under favorable folding conditions. Brownian dynamics simulations of a model polyelectrolyte suggest that the kinetics of counterion-mediated collapse and the dimensions of the collapsed RNA chains depend on the structure of the counterion. The results are consistent with delocalized condensation of polyamines around the RNA. However, the effective charge of the counterions is lowered by their excluded volume. The stability of the folded RNA is enhanced when the spacing between amino groups matches the distance between adjacent phosphate groups. These results show how changes in intracellular polyamine concentrations could alter RNA folding pathways.

Inner-sphere complexes of divalent cations with single-stranded poly(rA) and poly(rU).

A combination of ultrasound velocimetry, density, and UV spectroscopy has been employed to study the hydration effects of binding of Mn(2+) and alkaline-earth cations to poly(rA) and poly(rU) single strands. The hydration effects, obtained from volume and compressibility measurements, are positive due to overlapping the hydration shells of interacting molecules and consequently releasing the water molecules to bulk state. The volume effects of the binding to poly(rA), calculated per mole of cations, range from 30.6 to 40.6 cm(3) mol(-1) and the compressibility effects range from 59.2 x 10(-4) to 73.6 x 10(-4) cm(3) mol(-1) bar(-1). The volume and compressibility effects for poly(rU) are approximately 17 cm(3) mol(-1) and approximately 50 x 10(-4) cm(3) mol(-1) bar(-1), respectively. The comparative analysis of the dehydration effects suggests that the divalent cations bind to the polynucleotides in inner-sphere manner. In the case of poly(rU) the dehydration effects correspond to two direct coordination, probably between adjacent phosphate groups. The optical study did not reveal any effects of cation on the secondary structure or aggregation of poly(rU). In the case of single-helical poly(rA) binding is more specific: dehydration effects correspond to three to five direct contacts and must involve atomic groups of adenines, and the divalent cations stabilize and aggregate the polynucleotide.

Crystal structure of lithium cadmium diaqua catena-[monoborodiphosphate]- monohydrate, LiCd(H2O)2[BP2O8] · H2O

Article Crystal structure of lithium cadmium diaqua catena-[monoborodiphosphate]- monohydrate, LiCd(H2O)2[BP2O8] · H2O was published on December 1, 2003 in the journal Zeitschrift für Kristallographie - New Crystal Structures (volume 218, issue JG).

Binding of Mg 2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg 2+ to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 °C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg 2+ binding to the polynucleotides. Volume and compressibility effects of Mg 2+ binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg 2+ correspond to an inner-sphere complex with three–four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg 2+ on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg 2+ are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.

Enzyme Mechanism and Catalytic Property of β Propeller Phytase

Background: Phytases hydrolyze phytic acid ( myo-inositol-hexakisphosphate) to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the β propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. Results: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. Conclusions: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the “cleavage site”, which is responsible for the hydrolysis of a substrate, and the “affinity site”, which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo-inositol triphosphates from phytate and the hydrolysis of myo-inositol monophosphates.

Ribozyme catalysis via orbital steering

Orbital steering is invoked to explain how the three-dimensional structure of a small self-cleaving RNA, the hammerhead ribozyme, both prevents and enhances RNA autocatalysis. Within the conserved catalytic core of the ribozyme, the position of the 2′ oxygen atom of the G8 ribose is observed to be aligned almost perfectly with the phosphorus atom and the 5′ oxygen atom of the adjacent A9 phosphate group for self-cleavage via an in-line attack mechanism. Despite this apparent near-perfect atomic positioning, no cleavage takes place. The explanation proposed is that a network of hydrogen bonds in the ribozyme core orients or steers the orbitals containing the electron lone pairs of the attacking nucleophile (the 2′ oxygen atom) away from the A9 phosphorus atom, eliminating overlap with the vacant phosphorus d-orbitals despite the near-perfect in-line positioning of the oxygen atom, thus preventing catalysis. Because of the near-perfect atomic positioning of the 2′ oxygen atom relative to the phosphate group, orbital steering effects in this case are fortuitously uncoupled from conformational, distance and orientation effects, allowing an assessment of the catalytic power due purely to orbital steering. In contrast, a conformational change at the cleavage site is required to bring the 2′ oxygen atom and the scissile phosphate group into atomic positions amenable to an in-line attack mechanism. In addition, the conformationally changed structure must then steer the lone-pair orbitals of the correctly positioned 2′ oxygen atom toward the scissile phosphorus atom in order for cleavage to take place. We estimate that fulfilment of each of these two required changes may contribute separately an approximately 1000-fold rate enhancement, potentially accounting for a significant fraction of the catalytic power of this ribozyme. Orbital steering therefore appears to be a general phenomenon that may help to explain catalysis in both ribozymes and protein enzymes in a unified manner.

Hydrolysis of tRNAPhe on Suspensions of Amino Acids

RNA is adsorbed strongly on suspensions of many moderately soluble organic solids. In some cases, the hydrolysis of tRNAPhe is greatly accelerated by adsorption, and the major sites of hydrolysis are changed from those that are important in homogeneous solution. Here we show that the hydrolysis is greatly accelerated by suspensions of aspartic acid and β-glutamic acid but not by suspensions of α-glutamic acid, asparagine, or glutamine.

X-ray crystallographic observation of "in-line" and "adjacent" conformations in a bulged self-cleaving RNA/DNA hybrid.

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Hüsken et al., Biochemistry, 1996, 35:16591-16600). Transesterification proceeds via an in-line mechanism, with the 2'-OH of the bulged nucleotide attacking the 3'-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2'-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2'-oxygen of the bulged nucleotide and the P-O5' bond (including adjacent and near in-line) and give a detailed picture of the conformational changes necessary to line up the 2'-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308-1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.