Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P₋₁. Although RNase A also contains a P₋₁ subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P₋₁ subsite residues Arg³⁶, Asn³⁹, and Gln⁴⁰ of EDN in its catalytic activity. The individual mutation of Arg³⁶, Asn ³⁹, and Gln⁴⁰ resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P₋₁ subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates. Deepa Sikriwal and Divya Seth contributed equally to this work.     

Biochemical characterization of the RNA-hydrolytic activity of a pumpkin 2S albumin

A pumpkin 2S albumin with ribonuclease (RNase) activity was purified from pumpkin seeds (Cucurbita sp.) by liquid chromatographic techniques. It manifested potent RNase activity toward baker’s yeast RNA and calf liver RNA, and some polyhomoribonucleotides, including poly(A), poly(U) and poly(C) but not poly(G). Moreover, it was able to hydrolyze total RNA of both animal and plant origins. Ions such as Na⁺, Mg²⁺, Ca²⁺, and Zn²⁺ inhibited its RNase activity. Since RNase activity has not been previously reported in 2S albumins, this work may shed further light on the biological importance of this group of proteins.     

The presence of polyriboadenylic acid sequences in calf lens messenger RNA

The presence of poly(rA) sequences in lens RNA has been demonstrated by the isolation of RNase A and T₁-resistant fragments of approximately 50 nucleotide residues. These poly(rA)-rich sequences, obtained from lenses incubated for six hours in organ culture with [³H]adenosine, are located at the 3′ termini of mRNA as determined by 3′ exoribonuclease digestion. Limited digestion of the [³H]adenosine-labeled mRNA with the enzyme led to the abolition of binding to poly(rU)-filters and a concomitant loss of template activity with avian myeloblastosis virus RNA-dependent DNA polymerase. Furthermore, after incubation of lenses in organ culture with 3′-deoxyadenosine, the isolated polysomal RNA was unable to function as a template in an avian myeloblastosis virus RNA-dependent DNA polymerase-catalyzed reaction system.     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.     

A rapid method for mapping exposed cytosines in polyribonucleotides. Application to tRNATrp (yeast,beef liver)

A rapid method for mapping exposed cytosine residues in 5-[³²P]-labeled RNA molecules is suggested. The exposed cytosines (C's) are converted into uracyls (U's) by bisulphite treatment at pH 5.8 in the presence of Mg²⁺, followed by complete modification of the residual (non-exposed) C's by a methoxyamine and bisulphite mixture at pH 5.0. The control RNA is modified only by methoxyamine and bisulphite without the preliminary CU conversion. The location of the exposed C's is determined by comparing the products of partial T₁, T₂, A and U₂ ribonuclease digestions of the C U converted and control RNAs after slab gel polyacrylamide electrophoresis and autoradiography. The method has been applied for mapping exposed cytosine bases in tRNA^Trp (yeast) which have been found in the anticodon loop and at the 3-end of the molecule. In tRNA^Trp (beef liver), in addition to the same exposed bases, C in the diHU-loop is exposed. The data obtained are in full agreement with that is known about exposed C's for other tRNAs.     

Biological function of modified nucleotides T54 and Ψ55 of yeast tRNAAla

The 3′ half molecule of yeast tRNA^Ala (nucleotides 36–75) was hybridized with a DNA fragment (5′GGAATCGAACC 3′) and the hybrid was then digested withE. coli RNase H (from Boehringer). The enzyme can specifically cleave the 3′ half molecule at the 3′ side of nucleotide Ψ₅₅, thus a fragment C₃₆-Ψ₅₅ was prepared. The 3′-terminal T or TΨ of this fragment was removed by one or two cycles of periodate oxidation and β-elimination. The products were fragments C₃₆-T₅₄ and C₃₆-G₅₃. Three yeast tRNA_Ala fragments C₅₆-A₇₆, U₅₅-A₇₆ (with Ψ₅₅ replaced by U), U₅₄-A₇₆ (with T₅₄Ψ₅₅ replaced by UU) were synthesized and ligated with three prepared fragments (C₃₆-Ψ₅₅, C₃₆-T₅₄ and C₃₆-G₅₃) respectively by T4 RNA ligase. The products were further ligated with the 5′ half molecule (nu-cleotides 1–35). Using this method, one reconstituted yeast tRNA^Ala (tRNAr) and two yeast tRNA^ALa analogs: (i) tRNAa with U₅₅ instead of Ψ₅₅; (ii) tRNAb with U₅₄U₅₅ instead of T₅₄Ψ₅₅ were synthesized. The charging and incorporation activities of these three tRNAs were determined. In comparison with the reconstituted tRNA, the charging activity was 75% for tRNAa and 45% for tRNAb and the incorporation activity was 65% for tRNAa and 70% for tRNAb. These results suggest that the modified nucleotides T₅₄ and Ψ₅₅ play an important role in yeast tRNA^Ala function.     

Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA

Binding of Mn²⁺ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNA^Phe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNA^Phe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNA^Phe. Binding of Mn²⁺ to intact tRNA^Phe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn²⁺ to tRNA^Phe fragments (5′-$\text{1}\text{2}$, 3′-$\text{1}\text{2}$, 5′-$\text{3}\text{5}$, 3′-$\text{2}\text{5}$), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNA^Phe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections.     

Nucleotide sequences of two fragments from the coat-protein cistron of bacteriophage R17 ribonucleic acid

Bacteriophage R17 RNA was labelled with ³²P and was subjected to partial digestion with ribonuclease T₁. The products were fractionated by ionophoresis on polyacrylamide gel. Two fragments were purified and their nucleotide sequences determined by methods involving complete and further partial digestion with ribonucleases A and T₁. Fragment 20 had a sequence that coded for the amino acids in positions 32–53 of the coat protein of the bacteriophage. Fragment 20X, on further purification in 7m-urea, gave rise to two smaller nucleotides whose sequences coded for the amino acids in positions 56–66 and 67–76 of the coat protein. The sequence of the two fragments was such that they could be written in the form of loops stabilized by base-pairing.     

The effect of bisulfite modification on the template activity of DNA for DNA polymerase I

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO⁻₃)) residues by treatment with bisulfite. The poly(U(SO⁻₃)₂, C₃) and poly(U(SO⁻₃)₉, C₁) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO⁻₃) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO⁻₃) residues into U.     

Theory of melting of the triple helix poly (A + 2U) for a 1 : 2 mixture of Poly A to Poly U

The Zimm-Bragg theory is extended to treat the melting of the triple helix poly (A + 2U) for a solution with a 1 : 2 mole ratio of poly A to poly U. Only the case for long chains is considered. For a given set of parameters the theory predicts the fraction of segments in the triple helix, double helix, and random coil states as a function of temperature. Four nucleation parameters are introduced to describe the two order–disorder transitions (poly (A + 2U) ? poly A + 2 poly U and poly (A + U) ? poly A + poly U) and the single order–order transition (poly (A + 2U) ? poly (A + U) + poly U). A relation between the nucleation parameters is obtained which reduces the number of independent parameters to three. A method for determining these parameters from experiment is presented. From the previously published data of Blake, Massoulié and Fresco⁸ for [Na⁺] = 0.04, we find σ_T = 6.0 × 10^?4, σ_D = 1.0 × 10^?3, and σσ* = 1.5 × 10^?3. σ_T and σ_D are the nucleation parameters for nucleating a triple helix and double helix, respectively, from a random coil region. σσ* is the nucleation parameter for nucleating a triple helix from a double helix and a single strand. Melting curves are generated from the theory and compared with the experimental melting curves.     

Biological function of modified nucleotides T54 and Ψ55 of yeast tRNAAla

The 3′ half molecule of yeast tRNA^Ala (nucleotides 36–75) was hybridized with a DNA fragment (5′GGAATCGAACC 3′) and the hybrid was then digested withE. coli RNase H (from Boehringer). The enzyme can specifically cleave the 3′ half molecule at the 3′ side of nucleotide Ψ₅₅, thus a fragment C₃₆-Ψ₅₅ was prepared. The 3′-terminal T or TΨ of this fragment was removed by one or two cycles of periodate oxidation and β-elimination. The products were fragments C₃₆-T₅₄ and C₃₆-G₅₃. Three yeast tRNA_Ala fragments C₅₆-A₇₆, U₅₅-A₇₆ (with Ψ₅₅ replaced by U), U₅₄-A₇₆ (with T₅₄Ψ₅₅ replaced by UU) were synthesized and ligated with three prepared fragments (C₃₆-Ψ₅₅, C₃₆-T₅₄ and C₃₆-G₅₃) respectively by T4 RNA ligase. The products were further ligated with the 5′ half molecule (nu-cleotides 1–35). Using this method, one reconstituted yeast tRNA^Ala (tRNAr) and two yeast tRNA^ALa analogs: (i) tRNAa with U₅₅ instead of Ψ₅₅; (ii) tRNAb with U₅₄U₅₅ instead of T₅₄Ψ₅₅ were synthesized. The charging and incorporation activities of these three tRNAs were determined. In comparison with the reconstituted tRNA, the charging activity was 75% for tRNAa and 45% for tRNAb and the incorporation activity was 65% for tRNAa and 70% for tRNAb. These results suggest that the modified nucleotides T₅₄ and Ψ₅₅ play an important role in yeast tRNA^Ala function. Project supported by the National Natural Science Foundation of China.     

1H-nmr comparative studies of polynucleotides: Conformation and dynamic structure of polyribo(uridylic) and polyribo(cytidylic) acids in neutral solution

The conformation and the dynamic structure of single-stranded poly(U) and poly(C) in neutral aqueous solution have been investigated by ¹H-nmr at two different frequencies (90 and 250 MHz) and at various temperatures. Measurements of proton chemical shifts, coupling constants J_H-H, and proton relaxation times, T₁, T₂, versus temperature show a striking difference in conformation and in dynamic structure between the two polynucleotides studied. The temperature effect on δ and J_H-H is found to be substantial for poly(C) and insignificant for poly(U). The S conformer is favored in poly(U), whereas the N conformer strongly predominates in poly(C) (?90%), similar to the case for RNAs. These results suggest that single-stranded poly(C) probably possesses a helical or partial helical structure, whereas poly(U) shows a clear preference for the random coil, in agreement with the optical results. The local motions of the ribose and base were studied at various temperatures by measurements on the relaxation times at 90 and 250 MHz. For a given temperature between 22 and 72°C, the ratio T₁(90)/T₁(250) is practically the same for all poly(U) protons, indicating that in this temperature interval the ribose base unit of poly(U) undergoes an isotropic motion characterized by a single correlation time τ_c. Above 52°C, poly(C) exhibits a dynamic structure similar to poly(U). Below this temperature, poly(C) exists in an equilibrium between randomly coiled and single-stranded helix forms. This situation is characterized by a strong cross-relaxation effect and T₁ values corresponding to a relatively short apparent correlation time. An activation energy of 4 kcal/mol was determined for the motion of the ribose–base unit in both single-stranded polynucleotides.     

Unusual Contribution of 2-Aminoadenine to the Thermostability of DNA

Abstract

The poly(dA-dU) and poly(dl-dC) duplexes have very similar thermostabilities (T_m). This similarity extends also to the pyrimidine 5-methyl group-containing poly(dA-dT) and poly(dI-m⁵dC). The differences between chemical structures of the A:U and I:C or the A:T and I:m⁵C base-pairs seem to be unimportant for the thermostability of the DNA. However, on the insertion of an amino group into position 2 of the purines the similarities disappear. Thermostabilities of poly(n²dA-dU) and poly(dG-dC) as well as the poly(n²dA-dT) and poly(dG-m⁵dC) are radically different. This is also the case with their other 5-substituted pyrimidine-containing derivatives, the 5-ethyl, 5-n-butyl and 5-bromo analogues. The G:C-based polynucleotides are more stable by an average of 40°C than the n²A.U-based ones. Poly(dA,n²dA-dT)-s containing various proportions of A and n²A as well as the natural DNA of S-2L cyanophage that contains n²A bases instead of A were also studied. It was found that dependence of T_m on the n²A-content was non-linear and that the lower T_m is not the consequence of a particular nucleotide sequence. The possible structural reasons for the lower thermostabilization of these B-DNAs by the n²A:T base-pair as compared to the G:C are discussed.     

Formation of Hydrogen Peroxide by a Polyvinyl Alcohol Degrading Enzyme

A method for purification and crystallization, and some properties of ribonuclease from Aspergillus sp. [EC 2.7.7.17] (RNase L) are reported. The purification procedure consisted of six steps, including acetone precipitation, column chromatographies on Duolite A–2 and DEAE-cellulose, repeated chromatography on DEAE-Sephadex A–50 column and affinity chromatography on 5’-AMP-Sepharose 4B column. Crystallization was performed by the dialysis against ammonium sulfate solution at 60% saturation.

The crystalline enzyme was shown to be homogeneous by polyacrylamide disc electrophoresis and ultracentrifugation. Svedberg value of the crystalline enzyme was 4.2. The enzyme was the most active at pH 3.5 and 60~65°C, and it was inhibited markedly with Fe.³⁺

RNase L has no absolute base specificity, and produces four kinds of 3’-mononucleotides from yeast RNA. However, the susceptibility of four nucleotide residues to RNase L increases in the order; G < A < C < U and is quite different from those of RNase T₂, RNase M and RNase R.     

A common conformational feature in several prokaryotic and eukaryotic 5 S RNAs: a highly exposed, single-stranded loop around position 40

Psendomonas fluorescens, yeast and HeLa cells ³²P-labelled 5 S RNAs were submitted to partial hydrolysis with T₁, T₂ or pancreatic ribonucleases; the fragments were separated by two-dimensional acrylamide gel electrophoresis. First splits (obtained when only one cleavage takes place in the molecule) were found to occur essentially around position 40 in the sequence, as already demonstrated for Escherichia coli 5 S RNA. The existence in prokaryotic and eukaryotic 5 S RNAs of this very accessible region is thus proved. Eukaryotic 5 S RNAs also display a very accessible region around position 90 of the sequence.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg²⁺ or Mn²⁺) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T₁, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T₁ RNase.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.