Lead-catalysed specific cleavage of ribosomal RNAs.

Ribosomes have long been known to require divalent metal ions for their functional integrity. Pb2+-induced cleavage of the sugar-phosphate backbone has now been used to probe for metal binding sites in rRNA. Only three prominent Pb2+cleavages have been detected, with cleavage sites 5' of G240 in 16S rRNA and two sites 5' of A505 and C2347 in 23S rRNA. All cleavages occur in non-paired regions of the secondary structure models of the rRNAs and can be competed for by high concentrations of Mg2+, Mn2+, Ca2+ and Zn2+ ions, suggesting that lead is bound to general metal binding sites. Although Pb2+ cleavage is very efficient, ribosomes with fragmented RNAs are still functional in binding tRNA and in peptidyl transferase activity, indicating that the scissions do not significantly alter ribosomal structure. One of the lead cleavage sites (C2347 in 23S RNA) occurs in the vicinity of a region which is implicated in tRNA binding and peptidyl transferase activity. These results are discussed in the light of a recent model which proposes that peptide bond formation might be a metal-catalysed process.     

Complexity in orchestration of chemical groups near different cleavage sites in RNase P RNA mediated cleavage

RNase P mediated cleavage of the tRNA(His) precursor does not rely on the formation of the "+73/294 interaction" to give the correct cleavage product, i.e. cleavage at -1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the "2'OH-model", predicts that the 2'OH at the canonical cleavage site would affect cleavage at -1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNA(His) precursor cleavage site to investigate the role of 2'OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2'OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2'H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2'OH model. We favor a model where the 2'OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2'OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2'OH at different cleavage sites also indicated a higher dependence on the 2'OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position -1 in the substrate is most likely not involved in an interaction with RNase P RNA.     

Inhibition by lead ion of Electrophorus electroplax (Na+ + K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase.

Inorganic lead ion in micromolar concentrations inhibits Electrophorus electroplax microsomal (Na+ + K+)-adenosine triphosphatase ((Na+ + K+)-ATPase) and K+-p-nitrophenylphosphatase (NPPase). Under the same conditions, the same concentrations of PbCl2 that inhibit ATPase activity also stimulate the phosphorylation of electroplax microsomes in the absence of added Na+. Enzyme activity is protected from inhibition by increasing concentrations of microsomes, ATP, and other metal ion chelators. The kinetics follow the pattern of a reversible noncompetitive inhibitor. No kinetic evidence is elicited for interactions of Pb2+ with Na+, K+, Mg2+, ATP, or p-nitrophenylphosphate. Na+- ATPase, in the absence of K+, and (Na+ + K+)-NPPase activity at low [K+] are also inhibited. ATP inhibition of NPPase is not reversed by Pb2+. The calculated concentrations of free [Pb2+] that produce 50% inhibition are similar for ATPase and NPPase activities. Pb2+ may act at a single independent binding site to produce both stimulation of the kinase and inhibition of the phosphatase activities.     

Lead cleavage sites in the core structure of group I intron-RNA.

Self-splicing of group I introns requires divalent metal ions to promote catalysis as well as for the correct folding of the RNA. Lead cleavage has been used to probe the intron RNA for divalent metal ion binding sites. In the conserved core of the intron, only two sites of Pb2+ cleavage have been detected, which are located close to the substrate binding sites in the junction J8/7 and at the bulged nucleotide in the P7 stem. Both lead cleavages can be inhibited by high concentrations of Mg2+ and Mn2+ ions, suggesting that they displace Pb2+ ions from the binding sites. The RNA is protected from lead cleavage by 2'-deoxyGTP, a competitive inhibitor of splicing. The two major lead induced cleavages are both located in the conserved core of the intron and at phosphates, which had independently been demonstrated to interact with magnesium ions and to be essential for splicing. Thus, we suggest that the conditions required for lead cleavage occur mainly at those sites, where divalent ions bind that are functionally involved in catalysis. We propose lead cleavage analysis of functional RNA to be a useful tool for mapping functional magnesium ion binding sites.     

Cleavage of tRNA by Fe(II)-bleomycin.

We have investigated the action of the chemotherapeutic agent Fe(II)-bleomycin on yeast tRNA(Phe), an RNA of known three-dimensional structure. In the absence of Mg2+ ions, the RNA is cleaved preferentially at two major positions, A31 and G53, both of which are located at the terminal base pairs of hairpin loops, and coincide with the location of tight Mg2+ binding sites. A fragment of the tRNA (residues 47-76) containing the T stem-loop is also cleaved specifically at G53. Cleavage of both the intact tRNA and the tRNA fragment is abolished in the presence of physiological concentrations of Mg2+ (> 0.5 mM). Since Fe(II) is not displaced from bleomycin under these conditions, we infer that tight binding of Mg2+ to tRNA excludes productive interactions between Fe(II)-bleomycin and the RNA. These results also show that loss of cleavage is not due to Mg(2+)-dependent formation of tertiary interactions between the D and T loops. In contrast, cleavage of synthetic DNA analogs of the anticodon and T stem-loops is not detectably inhibited by Mg2+, even at concentrations as high as 50 mM. In addition, the site specificities observed in cleavage of RNA and DNA differ significantly. From these results, and from similar findings with other representative RNA molecules, we suggest that the cleavage of RNA by Fe(II)-bleomycin is unlikely to be important for its therapeutic action.     

Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination

We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function. We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.     

The role of magnesium ions in the regulation of the catalytic activity of kidney Na+,K+-ATPase

Free Mg2+ is studied for its effect on the activation kinetics of pig kidney Na+, K+-ATPase by monovalent cations (nH and K0.5 for Na+ and K+ are determined). It is established that at the saturating concentration of complementary ion-activator an increase of free Mg2+ concentration up to 12 mM is accompanied by a rise of nH and K0.5 for Na+ and a fall of K0.5 for K+ without nH changes for this cation. The analysis of inhibition kinetics shows that free Mg2+ is a competitive inhibitor as to Na+ and noncompetitive as to K+. It is concluded that inhibition of Na+, K+-ATPase by free Mg2+ is a complex process including competition with Na+ at its binding sites and the "occluding" of enzyme at the stage, preceding dissociation of cation and also the weakening of subunit interactions in the enzyme.     

In vitro selection of RNAs that undergo autolytic cleavage with Pb2+.

An in vitro selection method has been developed to obtain RNA molecules that specifically undergo autolytic cleavage reactions by Pb2+ ion. The method utilizes a circular RNA intermediate which is regenerated following the cleavage reaction to allow amplification and multiple cycles of selection. Pb2+ is known to catalyze a specific cleavage reaction between U17 and G18 of yeast tRNA(Phe). Starting from pools of RNA molecules which have a random distribution of sequences at nine or ten selected positions in the sequence of yeast tRNA(Phe), we have isolated many RNA molecules that undergo rapid and specific self-cleavage with Pb2+ at a variety of different sites. Terminal truncation experiments suggest that most of these self-cleaving RNA molecules do not fold like tRNA. However, two of the variants are cleaved rapidly with Pb2+ at U17 even though they lack the highly conserved nucleotides G18 and G19. Both specific mutations and terminal truncation experiments suggest that the D and T loops of these two variants interact in a manner similar to that of tRNA(Phe) despite the absence of the G18U55 and G19C56 tertiary interactions. A model for an alternate tertiary interaction involving a U17U55 pair is presented. This model may be relevant to the structure of about 100 mitochondrial tRNAs that also lack G18 and G19. The selection method presented here can be directly applied to isolate catalytic RNAs that undergo cleavage in the presence of other metal ions, modified nucleotides, or sequence-specific nucleases.     

Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.     

Uncoupling of ATP binding to Na+,K+-ATPase from its stimulation of ouabain binding: studies of the inhibition of Na+,K+-ATPase by a monoclonal antibody

The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.     

Expression of Na+,K+-ATPase in Pichia pastoris: analysis of wild type and D369N mutant proteins by Fe2+-catalyzed oxidative cleavage and molecular modeling

Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.     

Ethylenediamine as active site probe for Na+/K+-ATPase

(1) Ethylenediamine is an inhibitor of Na+- and K+-activated processes of Na+/K+-ATPase, i.e. the overall Na+/K+-ATPase activity, Na+-activated ATPase and K+-activated phosphatase activity, the Na+-activated phosphorylation and the Na+-free (amino-buffer associated) phosphorylation. (2) The I50 values (I50 is the concentration of inhibitor that half-maximally inhibits) increase with the concentration of the activating cations and the half-maximally activating cation concentrations (Km values) increase with the inhibitor concentration. (3) Ethylenediamine is competitive with Na+ in Na+-activated phosphorylation and with the amino-buffer (triallylamine) in Na+-free phosphorylation. Significant, though probably indirect, effects can also be noted on the affinity for Mg2+ and ATP, but these cannot account for the inhibition. (4) Inhibition parallels the dual protonated or positively charged ethylenediamine concentration (charge distance 3.7 A). (5) Direct investigation of interaction with activating cations (Na+, K+, Mg+, triallylamine) has been made via binding studies. All these cations drive ethylenediamine from the enzyme, but K+ and Mg+ with the highest efficiency and specificity. Ethylenediamine binding is ouabain-insensitive, however. (6) Ethylenediamine neither inhibits the transition to the phosphorylation enzyme conformation, nor does it affect the rate of dephosphorylation. Hence, we provisionally conclude that ethylenediamine inhibits the phosphoryl transfer between the ATP binding and phosphorylation site through occupation of cation activation sites, which are 3-4 A apart.     

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.     

D443 of the N domain of Na+,K+-ATPase interacts with the ATP-Mg2+ complex, possibly via a second Mg2+ ion

This paper provides evidence for an interaction of D443 in the N domain of Na(+),K(+)-ATPase with a Mg(2+) ion. Wild-type, D443N/A/C and S445A mutants of porcine Na(+),K(+)-ATPase (alpha1beta1) have been expressed in Pichia pastoris. By comparison with wild-type, D443N reduces the turn-over rate by about 40%. Binding affinity of ATP, measured directly, was not affected by D443N, D443A, or D443C mutations. AMP-PNP-Fe(2+)-catalyzed oxidative cleavage of Na(+),K(+)-ATPase produces two characteristic fragments, at (708)VNDS (P domain) and near (440)VAGDA (N domain), respectively. In the D443N and D443A mutants, both cleavages are suppressed, indicating an interaction between the residues with AMP-PNP-Fe(2+) bound. Previous work suggested that with ATP-Fe(2+) bound the N and P domains come into proximity, both D710 and D443 making contact with a single Fe(2+) (or Mg(2+)) ion. However, the crystal structure of Ca(2+)-ATPase with bound AMP-PCP and Mg(2+) confirm the involvement of D703 (D710) but show that E439 (D443) is too far to make contact with the Mg(2+). By contrast, in the crystal structure with bound ADP, AlF(4), and Mg(2+), representing the E(1)-P conformation, two Mg(2+) ions were observed. Significantly, ADP-Fe(2+)-mediated oxidative cleavage of renal Na,K-ATPase produces the fragment near (440)VAGDA (N domain), while the cleavage at (708)VNDS (P domain) is almost completely absent. The results are explained economically by the hypothesis that ATP is bound with two Mg(2+) (Fe(2+)) ions, a "catalytic" Mg(2+) interacting with D710 via the gamma phosphate and a "structural" Mg(2+) interacting with D443 via the alpha and beta phosphates and a water molecule, respectively.     

Effects of spermine and magnesium ions on the aminoacylation of yeast tRNA(Tyr)

Stimulatory effects of Mg2+ and spermine on the kinetics of the aminoacylation of tRNA(Tyr) were examined using purified yeast tRNA(Tyr) and tyrosyl-tRNA synthetase. The apparent Km for tRNA(Tyr) was the lowest at Mg2+ concentrations between 2 and 5 mM and was not influenced by spermine. In the absence of spermine, the apparent Vmax was the highest at Mg2+ concentrations of 5 mM or higher, whereas the presence of spermine strongly stimulated the reaction at lower Mg2+ concentrations. Spermine alone could not substitute for Mg2+, nor was it able, at any Mg2+ concentration, to increase the reaction rate above the level reached at high concentrations of Mg2+ alone. Calculations of the concentration of Mg3.tRNA(Tyr) complex as a function of initial Mg2+ concentration, using the binding constants derived from physical measurements, allow the conclusion that spermine exerts its stimulatory activity by creating strong binding sites for Mg2+; this would enable the tRNA to assume the conformation required for optimal aminoacylation. The conformational requirement for the first tRNA: synthetase encounter is obviously less stringent, since the apparent Km for tRNA(Tyr) is not influenced by spermine.     

Mg2+-induced tRNA folding

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.     

Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis.

A high yield, photoactivated cross-linking reaction between a modified tRNA and RNase P RNA was used as a quantitative assay of substrate binding affinity. The cross-linking assay allows the effects of metal ions on substrate binding to be measured independently and in the absence of the pre-tRNA cleavage reaction. The results of this assay, in conjunction with the conventional cleavage assay, support the following conclusions about the nature of the RNase P RNA-tRNA binding interaction. (i) Monovalent cations act primarily to enhance enzyme-substrate binding, presumably by functioning as counterions. This enhancement can be attributed to a reduction in the tRNA off-rate. (ii) Although divalent cation is required for cleavage, the enzyme-substrate complex can form in the absence of divalent cation; the essential role of divalent cation in the reaction is thus catalytic. (iii) Ca2+ is as efficient as Mg2+ in promoting binding but supports catalysis only at a low rate.     

Binding of spermidine to transfer ribonucleic acid

The binding of spermidine to yeast tRNAPhe and Escherichia coli tRNAGlu2 at low and high ionic strength was studied by equilibrium dialysis. Once corrected for the expected Donnan effect, the binding at low ionic strength obeys the simple relationship of equivalent binding sites, and cooperative binding of spermidine to tRNA could not be detected. At low ionic strength (0.013 M Na+ ion), tRNAPhe (yeast) has 13.9 +/- 2.3 strong spermidine binding sites per molecule with Kd = 1.39 X 10(-6) M and a few weak spermidine binding sites which were inaccessible to experimentation; tRNAGlu2 (E. coli) has 14.8 +/- 1.6 strong spermidine binding sites and 4.0 +/- 0.1 weak spermidine binding sites with Kd = 1.4 X 10(-6) M and Kd = 1.23 X 10(-4) M, respectively. At high ionic strength (0.12 M monovalent cation) and 0.01 M Mg2+, tRNAPhe (yeast) has approximately 13 strong spermidine binding sites with an apparent Kd = 3.4 X 10(-3) M while the dimeric complex tRNAPhe X tRNAGlu2 has 10.4 +/- 1.2 strong spermidine binding sites per monomer with an apparent Kd = 2.0 X 10(-3) M. In the presence of increasing Na+ ion or K+ ion concentration, spermidine binding data do not fit a model for competitive binding to tRNA by monovalent cations. Rather, analysis of binding data by the Debye-Hückel approximation results in a good fit of experimental data, indicating that monovalent cations form a counterion atmosphere about tRNA, thus decreasing electrostatic interactions. On the basis of equilibrium binding analyses, it is proposed that the binding of spermidine to tRNA occurs predominantly by electrostatic forces.     

tRNA(Phe) binds aminoglycoside antibiotics.

Aminoglycoside antibiotics have recently been found to bind to a variety of unrelated RNA molecules, including sequences that are important for retroviral replication. We report the binding of neomycin B, kanamycin A, and Neo-Neo (a synthetic neomycin-neomycin dimer) to tRNA(Phe). Using thermal denaturation studies, fluorescence spectroscopy, Pb2+-mediated tRNA(Phe) cleavage, and gel mobility shift assays, we have established that aminoglycosides interact with yeast tRNA(Phe) and are likely to induce a conformational change. Thermal denaturation studies revealed that aminoglycosides have a substantial stabilizing effect on tRNA(Phe) secondary and tertiary structures, much greater than the stabilization effect of spermine, an unstructured polyamine. Aminoglycoside-induced inhibition of Pb2+-mediated tRNA(Phe) cleavage yielded IC50 values of: 5 microM for Neo-Neo, 100 microM for neomycin B, > 1 mM for kanamycin A, and > 10 mM for spermine. Enzymatic and chemical footprinting indicate that the anticodon stem as well as the junction of the TpsiC and D loops are preferred aminoglycoside binding sites.     

Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity

The thermodynamics and kinetics of magnesium binding to tRNA(Phe)(yeast) have been studied directly by 25Mg NMR. In 0.17 M Na+(aq), tRNA(Phe) exists in its native conformation and the number of strong binding sites (Ka greater than or equal to 10(4)) was estimated to be 3-4 by titration experiments, in agreement with X-ray structural data for crystalline tRNA(Phe) (Jack et al., 1977). The set of weakly bound ions were in slow exchange and 25Mg NMR resonances were in the near-extreme-narrowing limit. The line shapes of the exchange-broadened magnesium resonance were indistinguishable from Lorentzian form. The number of weak magnesium binding sites was determined to be 50 +/- 8 in the native conformation and a total line-shape analysis of the exchange-broadened 25 Mg2+ NMR resonance gave an association constant Ka of (2.2 +/- 0.2) X 10(2) M-1, a quadrupolar coupling constant (chi B) of 0.84 MHz, an activation free energy (delta G*) of 12.8 +/- 0.2 kcal mol-1, and an off-rate (koff) of (2.5 +/- 0.4) X 10(3) s-1. In the absence of background Na+(aq), up to 12 +/- 2 magnesium ions bind cooperatively, and 73 +/- 10 additional weak binding sites were determined. The binding parameters in the nonnative conformation were Ka = (2.5 +/- 0.2) X 10(2) M-1, chi B = 0.64 MHz, delta G* = 13.1 +/- 0.2 kcal mol-1, and koff = (1.6 +/- 0.4) X 10(3) s-1. In comparison to Mg2+ binding to proteins (chi B typically ca. 1.1-1.6 MHz) the lower chi B values suggest a higher degree of symmetry for the ligand environment of Mg2+ bound to tRNA. A small number of specific weakly bound Mg2+ appear to be important for the change from a nonnative to a native conformation. Implications for interactions with the ribosome are discussed.