VPg unlinkase,the phosphodiesterase that hydrolyzes the bond between VPg and picornavirus RNA: a minimal nucleic moiety of the substrate

VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5"-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg–RNA complex were used: [¹²⁵I]Kp–pUpUpGp, [¹²⁵I]Kp–pUp, and [¹²⁵I]Kp–pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg–RNA). [¹²⁵I]K-peptides were unlinked from [¹²⁵I]Kp–pUpUpGp and [¹²⁵I]Kp–RNA with similar velocity, but [¹²⁵I]Kp–pUp was split much slower. Under the same conditions [¹²⁵I]Kp–pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [¹²⁵I]VPg–pU, [¹²⁵I]VPg–pUp, and [¹²⁵I]VPg–pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [¹²⁵I]VPg–RNA and [¹²⁵I]VPg–pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg²⁺. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH⁰ and ΔS⁰ for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH⁰ ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS⁰ ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg²⁺ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg²⁺, the ΔH⁰ and ΔS⁰ values are larger and temperature-dependent, with ΔH⁰ ≈ 22 kcal/mol (92 kJ/mol) and ΔS⁰ ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔC_p⁰ 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg²⁺ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg²⁺ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n_H) is 2.0 ± 0.1, indicative of the involvement of two Mg²⁺ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC₅₀ for the compound is 14 μM for the Mg²⁺-supported reaction, and 8 μM for the Mn²⁺-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.     

Reduced Model Captures Mg2+-RNA Interaction Free Energy of Riboswitches

The stability of RNA tertiary structures depends heavily on Mg²⁺. The Mg²⁺-RNA interaction free energy that stabilizes an RNA structure can be computed experimentally through fluorescence-based assays that measure Γ₂₊, the number of excess Mg²⁺ associated with an RNA molecule. Previous explicit-solvent simulations predict that the majority of excess Mg²⁺ ions interact closely and strongly with the RNA, unlike monovalent ions such as K⁺, suggesting that an explicit treatment of Mg²⁺ is important for capturing RNA dynamics. Here we present a reduced model that accurately reproduces the thermodynamics of Mg²⁺-RNA interactions. This model is able to characterize long-timescale RNA dynamics coupled to Mg²⁺ through the explicit representation of Mg²⁺ ions. KCl is described by Debye-Hückel screening and a Manning condensation parameter, which represents condensed K⁺ and models its competition with condensed Mg²⁺. The model contains one fitted parameter, the number of condensed K⁺ ions in the absence of Mg²⁺. Values of Γ₂₊ computed from molecular dynamics simulations using the model show excellent agreement with both experimental data on the adenine riboswitch and previous explicit-solvent simulations of the SAM-I riboswitch. This agreement confirms the thermodynamic accuracy of the model via the direct relation of Γ₂₊ to the Mg²⁺-RNA interaction free energy, and provides further support for the predictions from explicit-solvent calculations. This reduced model will be useful for future studies of the interplay between Mg²⁺ and RNA dynamics.     

A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg²⁺-μc's). Common features of Mg²⁺-μc's include two paired Mg²⁺ ions that are chelated by a common bridging phosphate group in the form Mg_(a)²⁺–(O1P-P-O2P)–Mg_(b)²⁺. This bridging phosphate is part of a 10-membered chelation ring in the form Mg_(a)²⁺–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg_(a)²⁺. The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg²⁺ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg²⁺-μc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg²⁺-μc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg²⁺-μc's in other rRNAs including the bacterial 16S rRNA, and the P4–P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg²⁺-μc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.     

Magnesium-binding architectures in RNA crystal structures: validation,binding preferences,classification and motif detection

The ubiquitous presence of magnesium ions in RNA has long been recognized as a key factor governing RNA folding, and is crucial for many diverse functions of RNA molecules. In this work, Mg²⁺-binding architectures in RNA were systematically studied using a database of RNA crystal structures from the Protein Data Bank (PDB). Due to the abundance of poorly modeled or incorrectly identified Mg²⁺ ions, the set of all sites was comprehensively validated and filtered to identify a benchmark dataset of 15 334 ‘reliable’ RNA-bound Mg²⁺ sites. The normalized frequencies by which specific RNA atoms coordinate Mg²⁺ were derived for both the inner and outer coordination spheres. A hierarchical classification system of Mg²⁺ sites in RNA structures was designed and applied to the benchmark dataset, yielding a set of 41 types of inner-sphere and 95 types of outer-sphere coordinating patterns. This classification system has also been applied to describe six previously reported Mg²⁺-binding motifs and detect them in new RNA structures. Investigation of the most populous site types resulted in the identification of seven novel Mg²⁺-binding motifs, and all RNA structures in the PDB were screened for the presence of these motifs.     

Application of Oligonucleoside Methylphosphonates in the Studies on Phosphodiester Hydrolysis by Serratia Endonuclease

Abstract

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg²⁺ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.     

Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3′p substrate to generate an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA^3′pp^5′A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA ^3′pp^5′A, the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3′p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in double-stranded DNA to produce a 3′-adenylated product. These 3′-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates.     

Multiforms of megamodulin-dependent protein kinases from baker's yeast

Summary Multiforms of megamodulin-dependent protein kinases (M-PK) were partially purified from baker's yeast by excluding endogenous megamodulin with histone, and then by gel filtration with Sephadex G-200. The stimulation of M-PK in the presence of Mg²⁺, Mn^2– or Co²⁺ was enhanced by yeast megamodulin. In addition, similar augmented activity of M-PK was also noted in the presence of Mg^2– by megamodulins prepared from E. coli, bovine brain and wheat germ.     

In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg²⁺ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.     

Filamentous growth ofPseudomonas aeruginosa

Summary The growth of two strains ofPseudomonas aeruginosa in stirred batch cultures was monitored by optical density, DNA concentration, and acridine orange direct cell count measurements. Growth of adherent bacteria in pure culture was also observed on suspended glass discs by light and scanning electron microscopy. Strain MUCOID produced significant numbers of filamentous cells in broth culture and in the adherent population, while strain PAO 381 did not produce elongated cells. Filamentous growth of MUCOID could be prevented by the addition of 5 × 10^–2 M Mg²⁺. However, the addition of 0.66 mM EDTA caused an increased proportion of the population (>50%) of MUCOID cells to become filamentous in broth culture. The results are discussed and related to theories regarding bacterial plasticity, and filamentation of normally bacillary cells.     

Production of RNA by a marine photosynthetic bacterium, Rhodovulum sp.

The marine photosynthetic bacterium, Rhodovulum sp. PS88, produces RNA not only in cells but also as an extracellular polymeric substance during aerobic continuous cultivation in the dark. At a dilution rate of 0.32–0.5 h^–1, the maximum RNA production was 460 mg RNA l^–1 broth (200 mg RNA g^–1 suspended solids) which is a value about 2–3 times more than that of yeast cells.     

Evidence for ditopic coordination of phosphate diesters to [Mg(15-crown-5)]<Superscript>2+</Superscript>. Implications for magnesium biocoordination chemistry

The interaction of a series of phosphate diesters and triesters (1=diphenyl phosphate, 2=dimethyl phosphate, 3=bis(2-ethylhexyl) phosphate, 4=trimethyl phosphate, 5=methyldiphenyl phosphate, 6=triphenyl phosphate) with [Mg(15-crown-5)]²⁺ (15-crown-5=1,4,7,10,13-pentaoxocyclopentadecane) was studied as a simplified model for the interaction of aqueous Mg²⁺ ion with phosphate-containing biomolecules such as RNA. Using electrospray mass spectrometry, we confirm the formation of 1:1 adducts in the gas phase. Proton and ³¹P NMR titration data were used to construct binding isotherms, and a 1:1 binding equilibrium was fit to the isotherms at room temperature to estimate the binding affinities. The binding affinity data are consistent with ditopic coordination of neutral dialkyl phosphate ligands to the [Mg(15-crown-5)]²⁺ unit. This involves inner-sphere coordination to the Mg²⁺ via an oxygen atom, which is complemented by a weak hydrogen-bonding interaction with the crown ether ligand. Ditopic interaction is consistent with low-temperature NMR spectra showing four different configurations for 1 coordinated to [Mg(15-crown-5)]²⁺, which are interpreted in terms of hindered rotation around the Mg–O_phos bond. Thermochemical analysis of the binding affinity data suggests that the second-shell interaction contributes only about 1 kcal/mol to the binding free energy, so additional factors, such as steric constraints, must be operative to give a preferred phosphate orientation in this system. However, the experimental data do suggest that second-shell interactions contribute as much as 40% of the total binding energy, consistent with the pronounced ability of aqueous Mg²⁺ to form salt-bridges linking secondary and tertiary elements of RNA structure.Abbreviations OTf trifluoromethanesulfonate - ESI-MS electrospray mass spectrometry     

Crystal structure,mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5′ splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg²⁺ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg²⁺ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg²⁺•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. Overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.     

Effect of Cd and Hg on the Activity of Na/K-ATPase and Mg-ATPase Adsorbed on Polystyrene Microtiter Plates

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4°C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na⁺/K⁺-ATPase and Mg²⁺-ATPase, retained 70–80% activity after the adsorption. In addition, adsorption stabilizes Na⁺/K⁺-ATPase and Mg²⁺-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na⁺/K⁺-ATPase and Mg²⁺-ATPase and their sensitivity to and mechanism of Cd²⁺- or Hg²⁺-induced inhibition. The only exception is the “high affinity” Mg²⁺-ATPase moiety, whose affinity for ATP and sensitivity toward Cd²⁺ were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′-kinase, a central 2′,3′ phosphatase, and a C-terminal ligase. Here we report the crystal structure of the kinase domain of Clostridium thermocellum Pnkp bound to ATP•Mg²⁺ (substrate complex) and ADP•Mg²⁺ (product complex). The protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain. ATP is bound within a crescent-shaped groove formed by the P-loop (¹⁵GSSGSGKST²³) and an overlying helix-loop-helix “lid.” The α and β phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides; the γ phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg²⁺ bridge the ATP β and γ phosphates. The P-loop serine (Ser22) is the sole enzymic constituent of the octahedral metal coordination complex. Structure-guided mutational analysis underscored the essential contributions of Lys21 and Ser22 in the ATP donor site and Asp38 and Arg41 in the phosphoacceptor site. Our studies suggest a catalytic mechanism whereby Asp38 (as general base) activates the polynucleotide 5′-OH for its nucleophilic attack on the γ phosphorus and Lys21 and Mg²⁺ stabilize the transition state.