Protonated polynucleotides structures - 22.CD study of the acid-base titration of poly(dG).poly(dC)

The acid-base titration (pH 8 --> pH 2.5 --> pH 8) of eleven mixing curve samples of the poly(dG) plus poly(dC) system has been performed in 0.15 M NaCl. Upon protonation, poly(dG).poly(dC) gives rise to an acid complex, in various amounts according to the origin of the sample. We have established that the hysteresis of the acid-base titration is due to the non-reversible formation of an acid complex, and the liberation of the homopolymers at the end of the acid titration and during the base titration: the homopolymer mixtures remain stable up to pH 7. A 1G:1C stoichiometry appears to be the most probable for the acid complex, a 1G:2C stoichiometry, as found in poly(C(+)).poly(I).poly(C) or poly(C(+)).poly(G).poly(C), cannot be rejected. In the course of this study, evidence has been found that the structural consequences of protonation could be similar for both double stranded poly(dG).poly(dC) and G-C rich DNA's: 1) protonation starts near pH 6, dissociation of the acid complex of poly(dG).poly(dC) and of protonated DNA take place at pH 3; 2) the CD spectrum computed for the acid polymer complex displays a positive peak at 255 nm as found in the acid spectra of DNA's; 3) double stranded poly(dG).poly(dC) embedded in triple-stranded poly(dG).poly(dG).poly(dC) should be in the A-form and appears to be prevented from the proton induced conformational change. The neutral triple stranded poly(dG).poly(dG).poly(dC) appears therefore responsible, although indirectly, for the complexity and variability of the acid titration of poly(dG).poly(dC) samples.     

Polysaccharide-polynucleotide complexes. Part 32. Structural analysis of the curdlan/poly(cytidylic acid) complex with semiempirical molecular orbital calculations

Natural Curdlan adopts a right-handed 6(1) triple helix, in which the constituting glucan chains are underpinned with each other by the intermolecular hydrogen bonds. Curdlan can form a stoichiometric complex with polynucleotides [e.g., poly(cytidylic acid), poly(C)]. In this paper, we carried out the MOPAC (semiempirical molecular-orbital package) calculation to examine the molecular structure of the Curdlan/poly(C) complex. The calculation exhibited that two types of hydrogen bonds are formed between the Curdlan and the poly(C); the third nitrogen (N3) in cytosine forms a hydrogen bond with the second OH of one Curdlan chain, and the proton of N4 is interacting with the O2 of another Curdlan chain. In our model, the helix diameter of poly(C) is expanded from 11.0 to 15.3 A upon complexation. Despite such large conformational changes, the 6(1) helix structure of poly(C) was maintained even after the complexation. This fact is complementary to the experimental fact that the complexation does not change the band shape of the circular dichroism of poly(C). The chain length dependence of the reaction enthalpy indicated that the complexation becomes thermodynamically more favorable with the chain length increasing. This feature is also consistent with the experimental data.     

Cooperative nonenzymic base recognition--a study of interaction of oligoguanylic acid with poly C

The interaction between poly C and (Gp)_nG(n = 1,2) in dilute solution was investigated spectrophotometrically in 0.1M phosphate buffer pH 7.2 under conditions unfavorable for the formation of self-associated complexes of oligoguanylic acids. Two isosbestic points were observed when poly C was titrated gradually with GpGpG, one at 232–233 mμ(in the range of 0–33% poly C) and one around 238 mμ (in the range of 50–100% poly C). The melting temperature (T_m) of the 1:1 poly C: (Gp)nG complexes (n = 1,2) of varying concentration were determined. The equilibrium properties of the 1:1 complexes can be described by two interaction parameters, namely, (i) cooperative stacking interaction between the first nearest neighbor of the adsorbed oligomer, and (ii) intrinsic association constant of the adsorbed oligomer with its polymeric site, since the cooperative helix–coil transition particularly in the smaller oligonucleotide can be described by an “all or none” model. Based on such a model the enthalpy of stacking inteaction-dependent T_m values yielded directly the sum of the enthalpy of stacking interaction and of basepairing (which is dependent on the chain length of the oligomer) and the value of S, the stability constant of a G–C pair within a helix. The enthalpy of formation of G–C pair is then calculated as ?6.3 kcal/base pair either from the chain length dependent enthalpy term or from the temperature coefficient of S values. From the S value and the association constant of 1:1 GpGpGpC:GpCpCpC complex, other thermodynamic parameters such as nucleation parameter (β) and free energy of stacking interaction can be obtained.     

Complexes of polyriboguanylate with modified polyribocytidylate

It was established minimal length of continuous poly(C) sequence of poly(G).poly(C) complex required for effective interferon induction by investigation poly(G).poly(C,A) with different molar ratios of C:A varying from 10:1 to 90:1. The minimum length of the double-stranded sequence of the macromolecule complex poly(G).poly(C) is equal to at least 90-100 nucleotides. The effect of 5-halogen-polyribocytidilates on the properties of the complexes has been also investigated.     

Effects of membrane ribonuclease and 3'-nucleotidase on the digestion of polyuridylic acid by rat liver plasma membrane.

1. Fragments of isolated rat liver plasma membrane possess a ribonuclease activity which at pH 7.8 in the presence of 10 mM EDTA can digest polyuridylic acid (poly(U)) and polycytidylic acid (poly(C)) but not polyadenylic acid (poly(A)) and polyguanylic acid (poly(G)). Under these conditions, the membrane preparation does not degrade native or denatured DNA. 2. The products of the reaction with poly(U) (10 mM EDTA present) can be separated on DEAE-Sephadex into oligonucleotides of increasing chain length. Most of the products are di- to hexa-nucleotides which contain terminal 3'-phosphate groups. 3. When EDTA is not present (pH 7.8 or 8.8) the plasma membrane preparation degrades both poly(A) and poly(U). With poly(A) the product is all nucleoside while with poly(U) as substrate most of the product is nucleoside, but also some oligonucleotides are produced. 4. The ribonuclease releases acid soluble products very slowly from high concentrations of poly(U) (mg/ml). 5. Uridine trinucleotide with and without a terminal 3'-phosphate group is degraded by rat liver plasma membrane. The trinucleotide diphosphate is rapidly hydrolyzed to nucleoside while the trinucleotide itself is slowly digested and yields intermediate products, including nucleoside.     

Protonated polynucleotide structures. IX. Disproportionation of poly (G)-poly (C) in acid medium

The interaction between poly (G) and poly (C) was investigated in neutral and acid medium by optical methods. Three main points arise from this investigation. (1) The formation of poly (G)·poly (C) was complete only above an ionic strength of about 0.6M [Na⁺]. Lowering the ionic strength increased the amounts of free poly (G) and free poly (C) that could be detected. (2) When titrating towards acid pH values a transition took place which was characterized by potentiometry, mixing curves, and circular dichroism: a three-stranded poly (G)·poly (C)·poly (C⁺) complex was formed analogous to the transition observed for the acid titration of poly (I)·poly (C). (3) Even when the poly (G)·poly (C) complex was incompletely formed (at low ionic strength) in neutral medium all poly (C) entered the triple-stranded complex.     

Physical and coding properties of poly(5-methoxyuridylic) acid.

The synthesis of poly(mo5U) requires a high concentration (2.7 mg/ml) of polynucleotide phosphorylase as well as a long reaction time (48 h). The resulting polynucleotide has a chain length of approximately 100 nucleotides. It shows no indication of a stable secondary structure. When poly(mo5U) is mixed with poly(A), a triple-stranded complex poly(A) . 2poly(mo5U) is formed. This complex has a melting temperature of 68.5 +/- 0.5 degrees C at 150 mMNa+ and exhibits a hysteresis loop between melting and reformation of the complex having a delta Tm of 11.5 degrees C. Poly-5-methoxyuridylic acid stimulates the binding of Phe-tRNA to 70-S ribosomes but is inactive in directing poly(Phe) synthesis.     

Complex formation between ribosomal protein S1, oligo-and polynucleotides: chain length dependence and base specificity.

In order to examine the nature of the complex formation between the ribosomal protein S1 and nucleic acids three methods were used: Inhibition of the reaction of n-ethyl[2.3 14C]-maleimide with S1 by the addition of oligonucleotides; adsorption of the complexes to nitrocellulose filters; and equilibrium dialysis. The complex formation is Mg2+ dependent at low salt concentrations and becomes Mg2+ independent at an ionic strength greater than 90 mM. Oligouridylates of increasing chain length reach an optimal KA of 3-3-10(7) M-1 at a chain length of n=13-14. Protein S1 contains one binding site for long chain oligouridylates, such as U12, and the standard-free-energy change on binding caused by one Pu increment is 0.41 kcal/mol, when n varies between five and fourteen. Complex formation is insensitive to the capacity of the homopolynucleotide bases to form hydrogen bonds. Homopolynuceotides, however, showing a Tm less than 250 in the buffer system used show an increased affinity for S1 compared to poly(A) and poly(C) (Tm greater than 40 degrees). The data are discussed with respect to the proposed binding of protein S1 to the 3-terminal end of the 16S RNA.     

Comparative studies on polyguanylate polymerase and polyadenylate polymerase activities in the DNA-dependent RNA polymerase I fraction from cauliflower.

The properties of poly(G) polymerase and poly(A) polymerase activities in the DNA-dependent RNA polymerase [nucleosidetriphosphate: RNA nucleotidyltransferase EC 2.7.7.6] I fraction from cauliflower (Brassica oleracea var. botrytis) were comparatively investigated. The pH optimum, the effect of ionic strength, the effect of substrate concentration on the rate of synthesis, the effect of divalent metal ion concentration, and the time course of synthesis at different temperatures were all different for the three polymerase activities. The enzyme fraction preferentially utilized denatured DNA. Synthetic poly(C) and poly(U) were more effectively utillized for the synthesis of polyguanylate and polyadenylate, respectively. Further, it was found that poly(G) and poly(A) formed in vitro by the enzyme fraction had chain length of 25-28 and 84-89 nucleotides, respectively, and that poly (adenylate-gluanylate) chain was hardly formed when ATP and GTP were added together as substrates in the same reaction medium.     

Poly(dG).poly(dC) at neutral and alkaline pH: the formation of triple stranded poly(dG).poly(dG).poly(dC).

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.     

First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan

Schizophyllan is a natural beta-(1-->3)-D-glucan that exists as a triple helix in H(2)O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH >13). As we have already reported, when a homo-polynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophyllan/DMSO solution, and, subsequently, DMSO is exchanged for H(2)O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H(2)O. In this report, we show that the complexation can be accelerated when the solution pH is changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA.     

Polysaccharide-polynucleotide complexes. Part 7. Hydrogen-ion and salt concentration dependence of complexation between schizophyllan and single-stranded homo RNAs

Schizophyllan belongs to a beta-1,3-D-glucan family, which exists as a random coil in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. The schizophyllan single chain forms a complex with single-stranded homo RNAs in water/DMSO mixed solvents. Using circular dichroism, we studied the complexation and its stability as a function of apparent pH (pH(*)) in a mixed solvent system and as a function of the salt concentration. The complex is formed in the pH(*) range 6.5-10, and dissociated in the pH(*) range 4-6. Both poly(A) and poly(C) adopt a double strand in the pH(*) range 4-6 and a single strand in the pH(*) range 6.5-10. Therefore, the conformational change of each polynucleotide is responsible for dissociation/association of the complex, i.e., the single strand of the polynucleotides can form complexes, whereas the double one cannot. This result indicates that hydrogen bonding and similarity of the helix parameters are essential for the complex formation. The melting temperature of the complex reaches the maximum around 0.05 M of NaCl and KCl, and the value of the maximum temperature depends on the cation species.     

Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C,A) templates

Poly(C,A) random copolymer templates direct the oligomerization of 2-MeImpG (2-MeImpX is the 5'-phospho-2-methylimidazolide of the nucleoside X) and 2-MeImpU, resulting in the production of a variety of oligo (G,U)s. This reaction is less efficient than comparable reactions involving poly(C,U) or poly(C,G) templates. The efficiency of monomer incorporation into newly synthesized oligomers is lower for 2-MeImpU than 2-MeImpG, and cannot be improved by increasing the concentration of 2-MeImpU relative to 2-MeImpG. This suggests that RNA templates containing runs of consecutive adenine residues would not be suitable for use in a chemical self-replicating system. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phosphodiester-linkage isomerism. Oligomers up to about the 13-mer, with base composition Gn, Gn-1, U, and Gn-2, U2, have been identified.     

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Human granulocyte ribonuclease.

Human granulocytes contain an RNase which is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for the secondary phosphate esters of uridine 3′-phosphates. It has no action on uridine 2′: 3′-cyclic phosphates. Poly (A) and poly (G) are inert to its action. Its rate of hydrolysis of poly (C) is about 1% of that of poly (U). It differs from bovine pancreatic RNase and human serum RNase. Because of its unique specificity, this enzyme might serve as a biochemical marker in certain granulocyte disorders.     

Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Purification and properties of a ribonuclease in human urine that hydrolyses polycytidylic acid.

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylamide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is acidic and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21,500. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3'-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2': 3'-cyclic phosphate or uridine 2':3'-cyclic phosphate. Its rate of hydrolysis of poly (U) is less than 2% of that of poly C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U). It differs from bovine pancreatic Rnase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.     

Purification and properties of an endoribonuclease existing as a complex with inhibitor in rat liver cytosol

An endoribonuclease existing as a complex with inhibitor in the cytosol of rat liver has been purified about 128,000-fold after inactivation of the inhibitor with CdCl2. The enzyme had a molecular weight of 16,000 and produced 3'-CMP via 2',3'-cyclic phosphate of cytidine from poly(C). The breakdown of poly(U) by the enzyme was less than 5% of poly(C) breakdown. Poly(A) was not hydrolyzed by the enzyme. The enzyme had a pH optimum of 7.5-8, was heat-stable and had a Km of 952 micrograms yeast RNA and a Km of 198 micrograms poly(C) per ml. The maximal velocities for yeast RNA and poly(C) degradation were 3,970 A260/min/mg protein and 1,890 A260/min/mg protein, respectively. The enzyme was slightly stimulated by polyamines or monovalent and divalent cations except Mn2+, but was inhibited by nucleoside triphosphate, poly(G) and rat liver RNase inhibitor. Inhibition of the enzyme by rat liver RNase inhibitor was not prevented by monovalent and divalent cations or polyamines, although inhibition by poly(G) was prevented by these ions.     

Initiation of DNA synthesis by the calf thymus DNA polymerase-primase complex

The calf thymus DNA polymerase-alpha-primase complex purified by immunoaffinity chromatography catalyzes the synthesis of RNA initiators on phi X174 single-stranded viral DNA that are efficiently elongated by the DNA polymerase. Trace amounts of ATP and GTP are incorporated into products that are full length double-stranded circular DNAs. When synthetic polydeoxynucleotides are used as templates, initiation and DNA synthesis occurs with both poly(dT) and poly(dC), but neither initiation nor DNA synthesis was observed with poly(dA) and poly(dI) templates. Nitrocellulose filter binding and sucrose gradient centrifugation studies show that the DNA polymerase-primase complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Using d(pA)-50 with 3'-oligo(dC) tails and d(pI)-50 with 3'-oligo(dT) tails, initiator synthesis and incorporation of deoxynucleotide can be demonstrated when the average pyrimidine sequence lengths are 8 and 4, respectively. These results suggest that purine polydeoxynucleotides are used as templates by the DNA polymerase only after initiation has occurred on the oligodeoxypyrimidine sequence and that the pyrimidine stretch required by the primase activity is relatively short. Analysis of initiator chain length with poly(dC) as template showed a series of oligo(G) initiators of 19-27 nucleotides in the absence of dGTP, and 5-13 nucleotides in the presence of dGTP. The chain length of initiators synthesized by the complex when poly(dT) or oligodeoxythymidylate-tailed poly(dI) was used can be as short as a dinucleotide. Analysis of the products of replication of oligo(dC)-tailed poly(dA) shows that initiator with chain length as low as 4 can be used for initiation by the polymerase-primase complex.     

Physical and chemical characterization of purified ovalbumin messenger RNA.

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.