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E Darzynkiewicz

Publications and source records attributed to E Darzynkiewicz.

At least 37 records · Page 2Linked to original sources
PubMed1990

A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E.

The binding of N-7-substituted cap analogues to eIF-4E from human erythrocytes is described. Data presented here indicate that there is a correlation between the tightness of binding of these cap analogues to eIF-4E and their potency as inhibitors of protein synthesis. This result indicates that the inhibitory activity of the cap analogues is strictly a function of the affinity of the analogue for eIF-4E under equilibrium conditions. The pH dependence of binding of the cap analogues to eIF-4E indicates that the enolate form of the cap is preferred, as originally postulated by Rhoads et al. [(1983) Biochemistry 22, 6084-6088]. Data indicate that there are differences in the mode of binding of alkyl-substituted and aryl-substituted cap analogues to eIF-4E arising from favorable interactions of the phenyl ring with the guanosine moiety. These differences may explain the enhanced recognition of the aryl-substituted cap analogues by eIF-4E.

Cross-Linking Reagents↗
PubMed1989

Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogues of mRNA 5'-cap: changes in N7 substituent affect analogue activity.

Nucleotide cap analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized in which the 7-methyl moiety was replaced with 7-ethyl (e7), 7-propyl (p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl (cp7), 7-(carboxymethyl) (cm7), 7-benzyl (bn7), 7-(2-phenylethyl) [7-(2-PhEt)], and 7-(1-phenylethyl) [7-(1-PhEt)]. These derivatives were assayed as competitive inhibitors of capped mRNA translation in reticulocyte lysate. We observed that N7 alkyl and alicyclic substituents larger than ethyl significantly decreased the inhibitory activity of these cap analogues presumably by decreasing their affinity for cap binding proteins, which participate in the initiation of translation. This result defined a maximum size for this class of N7 substituents in the nucleotide binding domain of cap binding proteins. Like m7GMP, the N7-substituted GMP derivatives synthesized in this study were found to be predominantly in the anti conformation as determined by proton NMR analyses. However, bn7GMP and 7-(2-PhEt)GMP, which have aromatic N7 substituents, were more effective than m7GMP as competitive inhibitors of translation. The increased affinity of bn7GMP for cap binding proteins was further examined by synthesis of beta-globin mRNA containing 5'-bn7G, 5'-m7G, or 5'-e7G cap structures. These modified mRNAs were tested as translation templates. Messenger RNA capped with bn7G was observed to increase the translation activity of the template 1.8-fold relative to that of its m7G-capped mRNA counterpart. By contrast, e7G-capped mRNA was 25% less active than m7G-capped mRNA.2+V photo-cross-linking of m7G-capped mRNA to cap binding proteins

Animals↗
PubMed1989

Base stacking of simple mRNA cap analogues. Association of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole and purine derivatives in aqueous solution.

Equilibrium constants for the association of different ionic forms of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole, caffeine and various methylated adenines have been determined by distributing the latter compounds between an organic solvent and aqueous solutions of the 7-methylguanine derivatives. The data are compared to those obtained for the association of unsubstituted purine with the same cosolutes. The stacking affinity of both cationic and zwitterionic forms of the 7-methylguanine ring correlates with the ring polarizability rather than the polarizing power of the cosolute. The cationic species stacks usually more efficiently. The chemical nature of the N9-substituent has only a moderate influence on the base-stacking properties.

Caffeine↗
PubMed1988

Beta-globin mRNAs capped with m7G, m2.7(2)G or m2.2.7(3)G differ in intrinsic translation efficiency.

We examined the intramolecular effect of altered cap structures on translation efficiency of artificial beta-globin mRNAs. For these studies, synthetic dinucleotides of the form X(5')ppp(5')G [X = 7-methyl guanosine (m7G), 2,7-dimethyl guanosine (m2(2,7)G) or 2,2,7-trimethyl guanosine (m3(2,2,7)G)], were transcriptionally incorporated into mRNAs, containing rabbit beta-globin coding sequences, using T7 RNA polymerase and a beta-globin cDNA template. These synthetic mRNAs were assayed in reticulocyte lysate for activity relative to m7G-capped mRNA. m2(2,7)G-Capped mRNA was found to be 1.5-fold more active than m7G-capped mRNA. Messenger RNA capped with m3(2,2,7)G was less active with activity of 0.24 relative to its m7G-capped counterpart (activity = 1.0). These data suggest that m7G-capped mRNAs become more active as translation templates after addition of a single N2 methyl moiety, which is especially pertinent to gene expression in togaviridae. The latter are observed to synthesize m2(2,7)G and m3(2,2,7)G-capped mRNAs in addition to m7G-capped templates during the course of infection in animal cells.

Animals↗
PubMed1988

Purine nucleoside phosphorylase. Structure-activity relationships for substrate and inhibitor properties of N-1-, N-7-, and C-8-substituted analogues; differentiation of mammalian and bacterial enzymes with N-1-methylinosine and guanosine.

The previous finding that 7-methylinosine (m7Ino) and 7-methylguanosine (m7Guo) are excellent, as well as fluorescent, substrates for calf spleen purine nucleoside phosphorylase has been extended to include a series of 7-alkylguanosines with higher alkyl groups (ethyl, propyl, butyl, isopropyl, isobutyl, benzyl). All of these are good substrates, with increases in Km compensated for by corresponding increases in Vmax, and excluding the ring N-7 as a binding site. Both m1Ino and m1Guo are neither substrates nor inhibitors of this enzyme, implicating the ring N-1 as a binding site. Included also are guanosine analogues with C-8 substituents (alpha-hydroxyisopropyl, methyl, bromo, chloro, amino) with known populations of syn and anti conformers about the glycosidic bond; while these did not unequivocally point to involvement of only one form, efficiency of phosphorolysis appeared to be correlated with a conformation in the anti region. The influence of various substituents is also considered in relation to steric and electronic effects. Kinetic parameters for all the foregoing have been determined fluorimetrically and/or spectrophotometrically from initial velocities and/or continuous monitoring of phosphorolysis including Ki values for inhibition by the base moieties. Furthermore, kinetic data demonstrated that, in striking contrast to the mammalian enzyme, both m1Ino and m1Guo are almost as good substrates as the parent nucleosides for a bacterial (Escherichia coli) enzyme, suggesting that the ring N-1 is not a binding site for the latter.

Animals↗
PubMed1988

7-Methylguanine nucleotides and their structural analogues; protolytic equilibria, complexing with magnesium (II) ion and kinetics for alkaline opening of the imidazole ring.

First-order rate constants for the alkaline opening of the imidazole ring of several 7-methylguanine nucleotides and their structural analogues were determined. The results obtained suggested that intramolecular interaction between the negatively charged 5'-phosphate group and the positively charged imidazole ring markedly retard the attack of hydroxide ion on the C8 atom of the 7-methylguanine ring. In contrast, hardly any influence on the acidities of the interacting base and phosphate moieties was detectable. No effect on the complexing of the phosphate group with magnesium(II) ion could be detected.

Chemical Phenomena↗
PubMed1987

Inhibition of eukaryotic translation by analogues of messenger RNA 5'-cap: chemical and biological consequences of 5'-phosphate modifications of 7-methylguanosine 5'-monophosphate.

New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of -O with -H, -CH3, or -NH2. Additional analogues were synthesized with 8-methyl- or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 1H and 31P NMR for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by -H and -CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by -NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the alpha-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation.

Animals↗
PubMed1987

Properties of 7-alkylguanosines as substrates of purine nucleoside phosphorylase.

A series of 7-alkyl analogues of guanosine was prepared by alkylation of 5'-GMP, and enzymatic dephosphorylation of the products to the corresponding nucleosides. The latter were all excellent, as well as fluorescent, substrates of calf spleen nucleoside phosphorylase. Kinetic parameters demonstrated that the purine ring N(7) is not a binding site for the enzyme.

Alkylation↗
PubMed1986

Reactions of pyrimidine nucleosides with aqueous alkalies: kinetics and mechanisms.

Kinetics for the reactions of various cytosine and uracil nucleosides and their alkyl derivatives with aqueous sodium hydroxide have been studied by liquid chromatography. Blocking of the glycosyl hydroxyl groups with alkyl groups and changes in the glycon moiety configuration have been observed to exert only moderate effects on the rate of deamination of cytosine nucleosides. Methylation of the 4-amino group retards deamination considerably, while a methyl substituent at C5 is rate accelerating and at C6 only moderately rate retarding. These findings have been accounted for by a mechanism involving a rate limiting bimolecular displacement of the 4-amino group by a hydroxide ion. Analogous comparisons with uracil nucleosides suggest that the decomposition of uridine is initiated by an intermolecular attack of hydroxide ion on the C5 atom of the base moiety. In contrast, beta-D-arabino- and beta-D-lyxo-furanosyl derivatives appear to be cleaved via an intramolecular nucleophilic attack of the ionized 2'-hydroxyl group.

Alkalies↗
PubMed1980

Base-pairing in conserved 3' end of 18 S rRNA as determined by psoralen photoreaction and RNase sensitivity.

Wheat and rabbit 18 rRNAs radiolabeled at the 3' end with [5'-32P]pCp were analyzed by polyacrylamide gel electrophoresis after chemical cleavage or partial enzymatic digestion under nondenaturing conditions. rRNA was similarly analyzed after photoreaction with 4'-aminomethyl-4,5',8-trimethylpsoralen and photoreversal of the resulting pyrimidine cross-links. The digestion patterns indicated the presence of a base-paired structure within a conserved 3'-terminal sequence. This structure corresponds to the 3'-proximal hairpin configuration suggested previously for the small subunit rRNAs of Escherichia coli, yeast, rat, and silkworm on the basis of their nucleotide sequences.

Animals↗
PubMed1980

Assignment of reovirus mRNA ribosome binding sites to virion genome segments by nucleotide sequence analyses.

All ten reovirus genome RNA segments were radiolabeled at their 3'-termini by incubation with RNA ligase and 32pCp. The extent of radiolabeling was similar for each of the double-stranded RNAs in the genome segment mixture. Radioactivity was equally distributed between the separated plus and minus strands indicating that the 5'-cap in plus strands did not block 3'-end-labeling of minus strands. The 3'-termini of the four S and three M segments included the common sequences: ...U-A-G-C in minus strands and ...U-C-A-U-C in plus strands. By comparing the minus strand 3'-sequences with 5'-sequences of reovirus mRNAs, small-size genome segments S2, S3 and S4 were correlated with the previously sequenced initiation fragments s46, s45 and s54 derived from small class mRNAs. Medium-size genome segments M1, M2 and M3 similarly were correlated with fragments m30, m52 and m44, respectively. The N-terminal amino acid sequences deduced from the mRNA nucleotide sequences can now be assigned to the nascent chains of particular reovirus proteins.

Base Sequence↗
PubMed1979

Correlations of conformational parameters and equilibrium conformational states in a variety of beta-D-arabinonucleosides and their analogues.

H nuclear magnetic resonance spectroscopy has been applied to a study of the conformations of a variety of purine and pyrimidine beta-D-arabinofuranosyl nucleosides. The experimental results, together with data collected from the literature, demonstrated the existence of reasonably good correlations between the coupling constants made it possible to define more accurately, than hitherto possible, the conformational states between which equilibria exist in solution. The equilibrium for the arabinonucleosides differs from that previously established for ribonucleosides; in particular, structural modifications and solvent effects may appreciably modify the conformational states between which equilibria exist. Preliminary measurements on some arabinosides in the syn conformation about the glycosidic bond indicated that these do not conform to the foregoing correlations, and will require separate study. A correlation has also been established between the conformation of the arabinose ring and that of the exocyclic 5'-CH2OH group. For both purine and pyrimidine arabinonucleosides, the conformational state 3E of the arabinose ring coexists to some extent with a gauche-gauche conformation of the exocyclic 5'-CH2OH, as in the case of pyrimidine (but not purine) ribonucleosides. Application of the foregoing to some biological problems is described.

Arabinonucleosides↗
PubMed1977

Kinetics of deamination of cytosine nucleosides with etherified sugar hydroxyls.

The kinetics of deamination of derivatives of the therapeutically important 1-beta-D-arabinofuranosylcytosine with etherified (methylated) sugar hydroxyls has provided additional direct evidence for involvement of the 2'-hydroxyl in intramolecular catalysed deamination. In the case of 1-beta-D-lyxofuranosylcytosine, the kinetics of deamination of its 2'-O-methyl and 3'-O-methyl derivatives pointed to similar involvement of the "up" 2'-OH in intramolecular catalysed deamination. Participation by the 3'-OH, which is also in the "up" position, was excluded. A qualitative correlation was shown to exist between the electron density distributions on C(4) and C(6) of the cytosine rings in cytosine, 1-methylcytosine and cytidine, and their relative susceptibilities to deamination.

Cytidine↗
PubMed1977

Preparation of O'-METHYL derivatives of 9-beta-D-xylofuranosyladenine.

Treatment of the therapeutically important 9-beta-D-xylofuranosyladine in strongly alkaline medium with dimethyl sulphate led principally to etherification of sugar hydroxyls and, to a minor extent, to formation of products with a methylated exocyclic amino group. The various O'-methyl derivatives of xylofuranosyladenine were fractionated on a strongly basic ion exchange column, and isolated in pure form. Also isolated was 9-beta-D-xylofuranosyl-N6-methyladenine and its 2'-O-methyl derivative. The products were identified from their 1H NMR spectra, for which extensive data are tabulated. The susceptibilities of the various derivatives to calf intestinal adenosine deaminase were examined in relation to those of other adenine nucleosides; in particular, 5'-O-methylation led to total loss of substrate properties for the riboside, arabinoside and xyloside of adenine.

Adenosine↗