A 31P-NMR study of mono- and dimagnesium complexes of adenosine 5′-triphosphate and model systems

³¹P-NMR chemical shifts and spin-lattice relaxation times of ATP (adenosine 5′-triphosphate), ribose 5′-triphosphate and tripolyphosphate show closely similar behaviour in aqueous solution at pH 7.5 on titration with Mg²⁺. The results are interpreted in terms of formation of 1 : 1 and 2 : 1 (dimagnesium) complexes with Mg²⁺ bound exclusively to the triphosphate chain. Stability constants for these complexes are reported. It is suggested that the predominant form of the 1 : 1 complexes has Mg²⁺ bound in tridentate manner (via non-bridging oxygen) to the α, β and γ phosphorus atoms; whilst that of the 2 : 1 complexes has each Mg²⁺ bound in bidentate manner, one to the α and β, and the other to the β and γ, phosphorus positions.     

Metal ion-N7 coordination in a ribozyme branch domain by NMR

The N7 of purine nucleotides presents one of the most dominant metal ion binding sites in nucleic acids. However, the interactions between kinetically labile metal ions like Mg²⁺ and these nitrogen atoms are inherently difficult to observe in large RNAs. Rather than using the insensitive direct ¹⁵N detection, here we have used ²J-[¹H,¹⁵N]-HSQC (Heteronuclear Single Quantum Coherence) NMR experiments as a fast and efficient method to specifically observe and characterize such interactions within larger RNA constructs. Using the 27 nucleotides long branch domain of the yeast-mitochondrial group II intron ribozyme Sc.ai5γ as an example, we show that direct N7 coordination of a Mg²⁺ ion takes place in a tetraloop nucleotide. A second Mg²⁺ ion, located in the major groove at the catalytic branch site, coordinates mainly in an outer-sphere fashion to the highly conserved flanking GU wobble pairs but not to N7 of the sandwiched branch adenosine.     

Study of the mitochondrial phosphate carrier in the course of calcium phosphate accumulation: A requirement for Mg2+ and ADP of its sensitivity to thiol reagents

1. The accumulation of calcium phosphate driven by succinate oxidation is ADP-dependent. In its absence the accumulation stops after a short incubation time and the oxygen uptake is permanently stimulated. This uncoupled oxygen uptake is insensitive to the inhibitors of phosphate transport, like mersalyl and N-ethylmaleimide. When ADP plus Mg²⁺ are added to the medium, or when ADP is added in the initial presence of magnesium, the inhibitory action of the thiol reagents on oxygen uptake is re-established. ADP alone or Mg²⁺ alone are without any effect.2. Phosphate/phosphate exchange has been studied, in the absence of ADP, when calcium phosphate accumulation had stopped and oxygen uptake is uncoupled. Under these conditions the exchange process becomes insensitive to thiol reagents. Sensitivity is recovered solely in the presence of ADP plus Mg²⁺.3. When mitochondrial swelling is studied according to the method of Chappell, it also appears that the phosphate carrier loses it sensitivity to mersalyl in the absence of ADP, which confirms the data obtained with phosphate/phosphate exchange experiments. When ADP plus Mg²⁺ are added (or present), together with mersalyl, the action of the thiol inhibitor is recovered. ADP and magnesium are inactive separately. EGTA plus Mg²⁺ (but not EGTA plus ADP) may substitute for ADP plus Mg²⁺ in this process.4. A possible interaction between the magnesium binding site and the phosphate carrier is considered and discussed.     

Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR

Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1′ and C5′ with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg²⁺ ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly ¹³C/¹⁵N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive ¹³C–¹³C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules.     

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.     

Towards predicting Ca2+‐binding sites with different coordination numbers in proteins with atomic resolution

Ca²⁺‐binding sites in proteins exhibit a wide range of polygonal geometries that directly relate to an equally‐diverse set of biological functions. Although the highly‐conserved EF‐Hand motif has been studied extensively, non‐EF‐Hand sites exhibit much more structural diversity which has inhibited efforts to determine the precise location of Ca²⁺‐binding sites, especially for sites with few coordinating ligands. Previously, we established an algorithm capable of predicting Ca²⁺‐binding sites using graph theory to identify oxygen clusters comprised of four atoms lying on a sphere of specified radius, the center of which was the predicted calcium position. Here we describe a new algorithm, MUG (MUltiple Geometries), which predicts Ca²⁺‐binding sites in proteins with atomic resolution. After first identifying all the possible oxygen clusters by finding maximal cliques, a calcium center (CC) for each cluster, corresponding to the potential Ca²⁺ position, is located to maximally regularize the structure of the (cluster, CC) pair. The structure is then inspected by geometric filters. An unqualified (cluster, CC) pair is further handled by recursively removing oxygen atoms and relocating the CC until its structure is either qualified or contains fewer than four ligand atoms. Ligand coordination is then determined for qualified structures. This algorithm, which predicts both Ca²⁺ positions and ligand groups, has been shown to successfully predict over 90% of the documented Ca²⁺‐binding sites in three datasets of highly‐diversified protein structures with 0.22 to 0.49 Å accuracy. All multiple‐binding sites (i.e. sites with a single ligand atom associated with multiple calcium ions) were predicted, as were half of the low‐coordination sites (i.e. sites with less than four protein ligand atoms) and 14/16 cofactor‐coordinating sites. Additionally, this algorithm has the flexibility to incorporate surface water molecules and protein cofactors to further improve the prediction for low‐coordination and cofactor‐coordinating Ca²⁺‐binding sites. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Ligation of water to magnesium chelates of biological importance

Water binding to several Mg²⁺ chelates, ethylenediamine, ethylenediamine-N,N’-diacetate, porphyrin, chlorophyll a and bacteriochlorophyll a, to form five- and six-coordinate complexes is studied by means of density functional theory. The results obtained for magnesium chelates are compared with the properties of the respective aqua complexes and the influence of the permittivity of environment on the ligand binding energies is discussed. Although the most common coordination number of Mg²⁺ is six, in the tetrapyrrolic chelates it is reduced to five because the accommodation of the sixth water ligand results in no gain in energy. This is in line with the experimental observations made for coordination of chlorophylls in vivo. The binding between Mg²⁺ and water is mostly of electrostatic nature, which is supported by the finding that its energy is correlated both with the electron density of the chelator and with electrostatic potential determined at the ligand binding site.     

Influence of divalent magnesium ion on DNA: molecular dynamics simulation studies

A large amount of experimental evidence is available on the effect of magnesium ions on the structure and stability of DNA double helix. Less is known, however, on how these ions affect the stability and dynamics of the molecule. The static time average pictures from X-ray structures or the quantum chemical energy minimized structures lack understanding of the dynamic DNA–ion interaction. The present work addresses these questions by molecular dynamics simulation studies on two DNA duplexes and their interaction with magnesium ions. Results show typical B-DNA character with occasional excursions to deviated states. We detected expected stability of the duplexes in terms of backbone conformations and base pair parameter by the CHARMM-27 force field. Ion environment analysis shows that Mg²⁺ retains the coordination sphere throughout the simulation with a preference for major groove over minor. An extensive analysis of the influence of the Mg²⁺ ion shows no evidence of the popular predictions of groove width narrowing by dipositive metal ion. The major groove atoms show higher occupancy and residence time compared to minor groove for magnesium, where no such distinction is found for the charge neutralizing Na⁺ ions. The determining factor of Mg²⁺ ion’s choice in DNA binding site evolves as the steric hindrance faced by the bulky hexahydrated cation where wider major groove gets the preference. We have shown that in case of binding of Mg²⁺ to DNA non electrostatic contributions play a major role.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:5     

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on (13)C-2-glycerol and (13)C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in (13)C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3' and C5' carbon positions. Consequently the C1', C2' and C4' positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with (13)C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a (13)C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4' carbon position, such that the C2' and C3' positions suffer from multiplet splitting but the C5' position remains singlet and the C1' position shows a small amount of residual C1'-C2' coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with (13)C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5' position (~90%) that makes it particularly attractive for NMR applications involving CH(2)-TROSY modules without the need for decoupling the C4' carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems.     

Retention of Ca2+ by rat liver and rat heart mitochondria: Effect of phosphate,Mg2+, and NAD(P) redox state

Parallel measurements of Ca²⁺ uptake, oxygen consumption, endogenous Mg²⁺ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca²⁺ in the presence of phosphate than rat liver mitochondria. The extent of Mg²⁺ efflux and swelling induced by Ca²⁺ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg²⁺ as a consequence of Ca²⁺ recycling across the membrane as induced by phosphate. Exogenous Mg²⁺ protects liver mitochondria against the deleterious effects of Ca²⁺ by inhibiting a ruthenium red-insensitive Ca²⁺ efflux induced by phosphate. Phosphate does not induce recycling of Ca²⁺ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca²⁺ recycling. In heart mitochondria the recycling of Ca²⁺ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca²⁺ is subject to metabolic control. In addition it has been observed that Sr²⁺does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca²⁺ than the ruthenium red-sensitive influx transporter.     

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

How calcium inhibits the magnesium‐dependent kinase gsk3β: A molecular simulation study

Glycogen synthase kinase 3β (GSK3β) is a ubiquitous serine/threonine kinase that plays a pivotal role in many biological processes. GSK3β catalyzes the transfer of γ‐phosphate of ATP to the unique substrate Ser/Thr residues with the assistance of two natural activating cofactors Mg²⁺. Interestingly, the biological observation reveals that a non‐native Ca²⁺ ion can inhibit the GSK3β catalytic activity. Here, the inhibitory mechanism of GSK3β by the displacement of native Mg²⁺ at site 1 by Ca²⁺ was investigated by means of 80 ns comparative molecular dynamics (MD) simulations of the GSK3β···Mg²⁺‐2/ATP/ Mg²⁺‐1 and GSK3β···Mg²⁺‐2/ATP/Ca²⁺‐1 systems. MD simulation results revealed that using the AMBER point charge model force field for Mg²⁺ was more appropriate in the reproduction of the active site architectural characteristics of GSK3β than using the magnesium‐cationic dummy atom model force field. Compared with the native Mg²⁺ bound system, the misalignment of the critical triphosphate moiety of ATP, the erroneous coordination environments around the Mg²⁺ ion at site 2, and the rupture of the key hydrogen bond between the invariant Lys85 and the ATP O^β2 atom in the Ca²⁺ substituted system were observed in the MD simulation due to the Ca²⁺ ion in active site in order to achieve its preferred sevenfold coordination geometry, which adequately abolish the enzymatic activity. The obtained results are valuable in understanding the possible mechanism by why Ca²⁺ inhibits the GSK3β activity and also provide insights into the mechanism of Ca²⁺ inhibition in other structurally related protein kinases. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (K_m ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg²⁺ ion in a manner that allows it to assist in transition state stabilization. The Mg²⁺ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg²⁺·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg²⁺ coordination in the human cytosolic (H_c)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in H_cTrpRS with a yet-to-be determined role for Mg²⁺. Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to H_cTrpRS.     

Basolateral Mg2+ Extrusion via CNNM4 Mediates Transcellular Mg2+ Transport across Epithelia: A Mouse Model

Transcellular Mg²⁺ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg²⁺ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg²⁺ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg²⁺ by exchanging intracellular Mg²⁺ with extracellular Na⁺. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg²⁺ extrusion activity. These results demonstrate the crucial importance of Mg²⁺ extrusion by CNNM4 in organismal and topical regulation of magnesium.     

Modulation of TRPM6 and Na+/Mg2+ exchange in mammary epithelial cells in response to variations of magnesium availability

Mammary epithelial cells (HC11) chronically adapted to grow in a low‐magnesium (0.05 mM vs. 0.5 mM) or in a high‐magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non‐physiological magnesium availability. Magnesium influx was higher in low‐magnesium cells compared to control or high‐magnesium cells, whereas magnesium efflux was higher in high‐magnesium cells compared to control and low‐magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na⁺/Mg²⁺ exchange. Using a monoclonal antibody detecting a ～70 kDa protein associated with Na⁺/Mg²⁺ exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high‐magnesium cells > control cells > low‐magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low‐magnesium cells re‐added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na⁺/Mg²⁺ exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney. J. Cell. Physiol. 222: 374–381, 2010. © 2009 Wiley‐Liss, Inc.     

Dysregulation of Mg2+ homeostasis contributes to acquisition of cancer hallmarks

Derangement of magnesium homeostasis underlies the pathophysiology of many diseases, including cancer. Recent advances support the view that aberrant expression of Mg²⁺ channels and other Mg²⁺ homeostatic factors may affect many hallmarks of cancer. The seminal idea of magnesium as a key regulator of cell proliferation has been enriched by novel intriguing findings that link magnesium and Mg²⁺ transporters to distinctive and complementary capabilities that enable tumour growth and metastatic dissemination. In this review, we examine the evidence on the involvement of members from the TRPM, CNNM and SCL41 protein families in cancer progression, and discuss their potential as therapeutic targets.     

Asymmetry of <Superscript>13</Superscript>C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

An effect of magnesium adenosine 5'-triphosphate on the structure of azoferredoxin from Clostridium pasteurianum

Azoferredoxin from $\text{Clostridium}\text{pasteurianum}$ has been treated anaerobically with 65 fold excess of α,α′-dipyridyl in the presence and absence of various nucleotides. Under reduced conditions 1% of the iron of AzoFd is chelated by α,α′-dipyridyl between 1 minute and 1 hour. However, when ATP is added in the presence of Mg²⁺, 80% of the iron in azoferredoxin is chelated within an hour. This effect is reproducible and nonenzymatic. The lack of this effect with other purine and pyrimidine nucleotides demonstrates that it is specific for magnesium ATP. Treatment of azoferredoxin with 4 M urea or oxygen in the presence of α,α′-dipyridyl induces a similar effect. An ATP-induced change in the availability of the iron in azoferredoxin to the chelator, α,α′-dipyridyl, is evidence that a conformational change has occurred.     

Structural effects of modified ribonucleotides and magnesium in transfer RNAs

Modified nucleotides are ubiquitous and important to tRNA structure and function. To understand their effect on tRNA conformation, we performed a series of molecular dynamics simulations on yeast tRNA^Phe and tRNA_init, Escherichia coli tRNA_init and HIV tRNA^Lys. Simulations were performed with the wild type modified nucleotides, using the recently developed CHARMM compatible force field parameter set for modified nucleotides (J. Comput. Chem. 2016, 37, 896), or with the corresponding unmodified nucleotides, and in the presence or absence of Mg²⁺. Results showed a stabilizing effect associated with the presence of the modifications and Mg²⁺ for some important positions, such as modified guanosine in position 37 and dihydrouridines in 16/17 including both structural properties and base interactions. Some other modifications were also found to make subtle contributions to the structural properties of local domains. While we were not able to investigate the effect of adenosine 37 in tRNA_init and limitations were observed in the conformation of E. coli tRNA_init, the presence of the modified nucleotides and of Mg²⁺ better maintained the structural features and base interactions of the tRNA systems than in their absence indicating the utility of incorporating the modified nucleotides in simulations of tRNA and other RNAs.     

Specificity of lens initiator tRNA for N-terminal recognition

The optimal magnesium ion concentration for chain initiation in a cell-free system derived from bovine eye lens which synthesizes 4 classes of crystallins appears to be 5 mM. In the synthesis of -crystallin polypeptides which contain one internal methionine residue and the second one in N-terminal position, Met-RNA^fMet functions exclusively as initiator. On the other hand at 5 mM Mg²⁺ Met-tRNA^Met inserts its methionine into the internal position. However, at higher magnesium ion concentrations the initiator tRNA also donates methionine for chain elongation while at the same time the cell-free system loses its capacity to initiate new polypeptides.