RESUMO
Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-ï¢-D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-ï¢-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-ï¢ -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.
Assuntos
2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Escherichia coli/efeitos dos fármacos , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Tubercidina/análogos & derivados , Tubercidina/farmacologia , 2-Aminopurina/síntese química , Acetaldeído/análogos & derivados , Acetaldeído/química , Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/enzimologia , Pirimidinas/química , Tubercidina/síntese químicaRESUMO
Synthetic oligonucleotides targeting functional regions of the prokaryotic rRNA could be promising antimicrobial agents. Indeed, such oligonucleotides were proven to inhibit bacterial growth. 2'-O-methylated (2'-O-Me) oligoribonucleotides with a sequence complementary to the decoding site in 16S rRNA were reported as inhibitors of bacterial translation. However, the binding mode and structures of the formed complexes, as well as the level of selectivity of the oligonucleotides between the prokaryotic and eukaryotic target, were not determined. We have analyzed three 2'-O-Me oligoribonucleotides designed to hybridize with the models of the prokaryotic rRNA containing two neighboring aminoglycoside binding pockets. One pocket is the paromomycin/kanamycin binding site corresponding to the decoding site in the small ribosomal subunit and the other one is the close-by hygromycin B binding site whose dynamics has not been previously reported. Molecular dynamics (MD) simulations, as well as isothermal titration calorimetry, gel electrophoresis and spectroscopic studies have shown that the eukaryotic rRNA model is less conformationally stable (in terms of hydrogen bonds and stacking interactions) than the corresponding prokaryotic one. In MD simulations of the eukaryotic construct, the nucleotide U1498, which plays an important role in correct positioning of mRNA during translation, is flexible and spontaneously flips out into the solvent. In solution studies, the 2'-O-Me oligoribonucleotides did not interact with the double stranded rRNA models but all formed stable complexes with the single-stranded prokaryotic target. 2'-O-Me oligoribonucleotides with one and two mismatches bound less tightly to the eukaryotic target. This shows that at least three mismatches between the 2'-O-Me oligoribonucleotide and eukaryotic rRNA are required to ensure target selectivity. The results also suggest that, in the ribosome environment, the strand invasion is the preferred binding mode of 2'-O-Me oligoribonucleotides targeting the aminoglycoside binding sites in 16S rRNA.
Assuntos
Modelos Moleculares , Oligonucleotídeos/química , RNA Ribossômico 16S/química , Calorimetria , Eletroforese em Gel de Poliacrilamida , Espectrometria de FluorescênciaRESUMO
7-Methylguanosine (m(7)G) nucleotides labelled with acetylpyrene (AcPy) were synthesized as fluorescent mRNA 5' end (cap) analogues. The unique fluorescent properties of m(7)G-AcPy conjugates, different from G-AcPy, can be applied to studying various mRNA cap-related processes including the evaluation of putative inhibitors of DcpS enzyme-a therapeutic target in neuromuscular diseases.
Assuntos
Guanina/análogos & derivados , Nucleotídeos/química , Pirenos/química , Fluorescência , Guanina/química , Espectrometria de FluorescênciaRESUMO
Chemically modified analogues of nucleosides and nucleotides, have been thoroughly investigated since the discovery of DNA double helix by Watson and Crick in 1953 (Nature 171: 737). Chemical structures, first of all tautomerism, of the nucleic acid bases, as well as the conformations of the nucleic acids constituents, determine the secondary and tertiary structures of DNA and RNA polymers. Similarly, structural and dynamic parameters of nucleoside derivatives determine their biological activity in mutagenesis, neoplastic transformation, as well as antiviral or anticancer properties. In this review, a multidisciplinary approach of Prof. David Shugar's group is presented in the studies on nucleosides and nucleotides. It consists in chemical syntheses of suitable analogues, measurements of physicochemical and spectral parameters, conformational analysis by means of nuclear magnetic resonance (NMR) and X-ray diffraction, as well as characteristics of the nucleoside analogues as inhibitors of some selected, target enzymes, crucial in respect to antiviral activity of the analogues. These long-lasting studies follows upon the line of the main paradigm of molecular biophysics, i. e. structure-activity relationship.
Assuntos
Antivirais/história , Bioquímica/história , Nucleosídeos/história , Nucleotídeos/história , Antivirais/química , Antivirais/uso terapêutico , História do Século XX , História do Século XXI , Conformação Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Nucleosídeos/uso terapêutico , Nucleotídeos/síntese química , Nucleotídeos/química , Nucleotídeos/uso terapêutico , Polônia , Análise Espectral/históriaRESUMO
Initiation is the rate-limiting step during mRNA 5' cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Iniciação Traducional da Cadeia Peptídica/fisiologia , Fosfoproteínas/metabolismo , Multimerização Proteica/fisiologia , Capuzes de RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Ciclo Celular , Fator de Iniciação 4E em Eucariotos/química , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/metabolismo , Fracionamento por Campo e Fluxo , Humanos , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fosfoproteínas/química , Ligação Proteica/fisiologia , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , UltracentrifugaçãoRESUMO
Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m7G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Proteínas de Helminto/química , Iniciação Traducional da Cadeia Peptídica , Análogos de Capuz de RNA/química , Animais , Ascaris suum , Fosfatos de Dinucleosídeos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas de Helminto/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Líder para Processamento/químicaRESUMO
Molecular mechanisms underlying the recognition of the mRNA 5' terminal structure called "cap" by the eukaryotic initiation factor 4E (eIF4E) are crucial for cap-dependent translation. To gain a deeper insight into how the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van't Hoff analysis based on intrinsic protein fluorescence quenching upon titration with a series of chemical cap analogs were performed, providing a consistent thermodynamic description of the binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed similarities and differences between yeast and mammalian eIF4Es. The yeast eIF4E complex formation was enthalpy-driven and entropy-opposed for each cap analog at 293 K. A nontrivial isothermal enthalpyentropy compensation was found, described by a compensation temperature, T(c) = 411 ± 18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected, ΔC(p)° = +5.2 ± 1.3 kJ·mol(-1)·K(-1). The charge-related interactions involving the 5'-5' triphosphate bridge of the cap and basic amino acid side chains at the yeast eIF4E cap-binding site were significantly weaker (by ΔΔH°(vH) of about +10 kJ·mol(-1)) than those for the mammalian homologues, suggesting their optimization during the evolution.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Capuzes de RNA/química , RNA Mensageiro/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Difração de Raios XRESUMO
Specific recognition and binding of the ribonucleic acid 5' termini (mRNA 5' cap) by the eukaryotic translation initiation factor 4E (eIF4E) is a key, rate limiting step in translation initiation. Contrary to mammalian and yeast eIF4Es that discriminate in favor of 7-methylguanosine cap, three out of five eIF4E isoforms from the nematode Caenorhabditis elegans as well as eIF4Es from the parasites Schistosome mansoni and Ascaris suum, exhibit dual binding specificity for both 7-methylguanosine-and N(2),N(2),7-trimethylguanosine cap. To address the problem of the differences in the mechanism of the cap recognition by those highly homologic proteins, we carried out molecular dynamics simulations in water of three factors, IFE-3 and IFE-5 isoforms from C. elegans and murine eIF4E, in the apo form as well as in the complexes with 7-methyl-GDP and N(2),N(2),7-trimethyl-GDP. The results clearly pointed to a dynamical mechanism of discrimination between each type of the cap, viz. differences in mobility of the loops located at the entrance into the protein binding pockets during the cap association and dissociation. Additionally, our data showed that the hydrogen bond involving the N(2)-amino group of 7-methylguanosine and the carboxylate of glutamic acid was not stable. The dynamic mechanism proposed here differs from a typical, static one in that the differences in the protein-ligand binding specificity cannot be ascribed to formation and/or disruption of well defined stabilizing contacts.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Capuzes de RNA/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Metilação , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Alinhamento de SequênciaRESUMO
The heterodimeric nuclear cap-binding complex (CBC) specifically recognizes the monomethylguanosine 5' cap structure of the eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is essential for nuclear maturation of mRNA, for nuclear export of U snRNA in metazoans, and for nonsense-mediated decay of mRNA and the pioneer round of translation. We analysed the recognition of the cap by native human CBC and mutants in which each tyrosine that stacks with the 7-methylguanosine moiety was replaced by phenylalanine or alanine and both tyrosines were replaced by phenylalanines. The equilibrium association constants (K(as)) for two selected cap analogues, P(1)-7-methylguanosine-5' P(3)-guanosine-5' triphosphate and 7-methylguanosine triphosphate, were determined by two independent methods, fluorescence titration and surface plasmon resonance. We could distinguish two tyrosines, Y43 and Y20, in stabilization of the cap inside the CBC-binding pocket. In particular, lack of Y20 in CBC leads to a greater affinity of the mono- than the dinucleotide cap analogue, in contrast to the wild-type protein. A crucial role of cation-pi stacking in the mechanism of the specific cap recognition by CBC was postulated from the comparison of the experimentally derived Gibbs free binding energy (DeltaG degrees) with the stacking energy (DeltaE) of the 7-methylguanosine/Y binary and ternary complexes calculated by the Møller-Plesset second-order perturbation method. The resulting kinetic model of the association between the capped RNA and CBC, based on the experimental data and quantum calculations, is discussed with respect to the "CBC-to-eukaryotic initiation factor 4E handoff" of mRNA.
Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Capuzes de RNA/metabolismo , Tirosina/metabolismo , Substituição de Aminoácidos/genética , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Tirosina/genéticaRESUMO
Recognition of the ribonucleic acid 5' termini (RNA 5' cap) by a wide class of cap-binding proteins is largely accomplished by cation-pi stacking that involves the positively charged 7-methylguanine ring and aromatic amino acid side chains. Quantum calculations of the stacking energy were performed by means of MP2 perturbation method for binary and ternary associates composed of the 7-methylguanine moiety and tryptophan, tyrosine, or phenylalanine, in their spatial orientations known from the crystalline cap-protein complexes. The results clearly pointed to an enhancement of the stacking energy due to a net positive charge in the cap guanine moiety and allowed analysis of a role of various amino acids in stabilization of the complexes. Conformational flexibility of the aromatic amino acids taking part in binding ligands to a wide class of RNA-recognizing proteins, including the cap-binding proteins, was determined by regional order neural network (RONN) algorithm that provides results close to those of the crystallographic B-factors analysis. Interestingly, some of the tyrosines that are classified in general as "rigid" showed high flexibility when engaged in binding the cap to nuclear cap-binding protein complex CBC and to viral methyltransferase VP39. Parallel analyses of the binding energy and flexibility of the protein fragments engaged in the binding leads to understanding differences in molecular mechanisms of the cap recognition by various proteins, CBC compared with the eukaryotic initiation factor eIF4E, and enzymes vs. other protein factors.
Assuntos
Aminoácidos Aromáticos/química , Proteínas de Ligação ao Cap de RNA/química , Capuzes de RNA/química , Algoritmos , Cristalografia por Raios X , Guanosina/análogos & derivados , Guanosina/química , Ligantes , Metilação , Modelos Moleculares , Estrutura Molecular , Redes Neurais de Computação , Ligação Proteica , Estrutura Secundária de Proteína , Teoria Quântica , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Triptofano/química , Tirosina/química , Água/químicaRESUMO
Recognition of the 5' cap by the eukaryotic initiation factor 4E (eIF4E) is the rate-limiting step in the ribosome recruitment to mRNAs. The regular cap consists of 7-monomethylguanosine (MMG) linked by a 5'-5' triphosphate bridge to the first transcribed nucleoside, while some primitive eukaryotes possess a N (2), N (2),7-trimethylguanosine (TMG) cap structure as a result of trans splicing. Mammalian eIF4E is highly specific to the MMG form of the cap in terms of association constants and thermodynamic driving force. We have investigated conformational changes of eIF4E induced by interaction with two cap analogues, 7-methyl-GTP and N (2), N (2),7-trimethyl-GTP. Hydrogen-deuterium exchange and electrospray mass spectrometry were applied to probe local dynamics of murine eIF4E in the apo and cap-bound forms. The data show that the cap binding induces long-range conformational changes in the protein, not only in the cap-binding pocket but also in a distant region of the 4E-BP/eIF4G binding site. Formation of the complex with 7-methyl-GTP makes the eIF4E structure more compact, while binding of N (2), N (2),7-trimethyl-GTP leads to higher solvent accessibility of the protein backbone in comparison with the apo form. The results suggest that the additional double methylation at the N (2)-amino group of the cap causes sterical effects upon binding to mammalian eIF4E which influence the overall solution dynamics of the protein, thus precluding formation of a tight complex.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Guanosina/análogos & derivados , Análogos de Capuz de RNA/química , RNA Mensageiro/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Fator de Iniciação 4E em Eucariotos/metabolismo , Guanosina/química , Guanosina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Análogos de Capuz de RNA/metabolismo , Proteínas de Ligação ao Cap de RNA/química , Proteínas de Ligação ao Cap de RNA/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Enzymatic cleavage of the P-chiral diastereoisomers of the 5' mRNA cap analogue bearing phosphorothioate moiety in alfa position of 5',5'-triphosphate bridge (m(7)Gppp(S)G D1 and D2) was performed by human Decapping Scavenger (DcpS) enzyme. Analysis of the degradation products allowed to estimate the absolute configuration at the asymmetric phosphorus atoms in examined compounds via correlation with the R(P) and S(P) diastereoisomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS).
Assuntos
Endorribonucleases/química , Conformação de Ácido Nucleico , Análogos de Capuz de RNA/química , Capuzes de RNA/química , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/análise , Humanos , Hidrólise , Tionucleotídeos/análiseRESUMO
For deeper understanding the roles of the mRNA cap structure in cellular processes isotopically labeled dinucleotide cap analogues have been synthesized as tools for NMR and in vivo studies. Tritium or carbon C-13 labeled methyl iodide was used as a source of the isotope material. In order to minimize the number of steps during the radioisotopic synthesis the methylation with tritium labeled methyl iodide was performed with Gp(3)G as a substrate. The C-13 isotope was introduced into the cap dinucleotide by methylation of GDP with C-13 Methyl iodide, followed by coupling the product with guanosine 5'-phosphorimidazolide in DMF with zinc chloride as a catalyst.
Assuntos
Fosfatos de Dinucleosídeos/química , Marcação por Isótopo , Ressonância Magnética Nuclear Biomolecular , Capuzes de RNA/química , RNA Mensageiro/química , Isótopos de Carbono/química , Catálise , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/química , Hidrocarbonetos Iodados/química , Metilação , Trítio/químicaRESUMO
Synthesis of Leishmania mRNA 5'-cap analogs, m(7)Gpppm(2)(6)AmpAm (cap-2), and m(7)Gpppm(2)(6)AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4).
Assuntos
Guanosina Pentafosfato/análogos & derivados , Guanosina Pentafosfato/síntese química , Leishmania/metabolismo , Análogos de Capuz de RNA/síntese química , Capuzes de RNA/síntese química , RNA de Protozoário/síntese química , Animais , Fator de Iniciação 4E em Eucariotos/química , Guanosina Pentafosfato/química , Análogos de Capuz de RNA/química , Capuzes de RNA/química , RNA de Protozoário/químicaRESUMO
Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m(7)Gp(3)G have found a wide application in studying such processes as mRNA translation, pre-mRNA splicing, mRNA turnover, and intracellular transport of mRNA and snRNA. However, because of the presence of a 3'-OH on both m(7)Guo and Guo moieties of the cap structure, one-third to one-half of the mRNAs contain a cap incorporated in the reverse orientation. The reverse cap structures bind poorly to eIF4E, the cap binding protein, and reduce overall translational efficiency. We therefore replaced the conventional m(7)Gp(3)G cap by "anti-reverse" cap analogs (ARCAs) in which the 3'-OH of m(7)Guo moiety was substituted by 3'-deoxy or 3'-O-methyl groups, leading to m(7)3'dGp(3)G or m(2)(7,3'-O) Gp(3)G, respectively. The class of ARCAs was extended to analogs possessing an O-methyl group or deoxy group at C2' of m(7)Guo. We have also developed a series of ARCAs containing tetra- and pentaphosphates. mRNAs capped with various ARCAs were translated 1.1- to 2.6-fold more efficiently than their counterparts capped with m(7)Gp(3)G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the alpha- and beta-, or beta- and gamma-phosphate moieties, leading to m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m(2)(7,3'-O)Gpp(CH2)pG were more stable when introduced into cultured mammalian cells. In this chapter, we describe the synthesis of representative ARCAs and their biophysical and biochemical characterization, with emphasis on practical applications in mRNA translation.
Assuntos
Biossíntese de Proteínas , Análogos de Capuz de RNA/síntese química , Estabilidade de RNA , Animais , Sistema Livre de Células/metabolismo , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Metano/análogos & derivados , Metano/química , Modelos Biológicos , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Especificidade por Substrato , Transcrição GênicaRESUMO
Specific recognition of mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Structural determination of the eIF4E-cap complexes, as well as complexes of eIF4E with other proteins regulating its activity, requires complementary experiments that allow for energetic and dynamic aspects of formation and stability of the complexes. Such a combined approach provides information on the binding mechanisms and, hence, may lead to mechanistic models of eIF4E functioning and regulation on the molecular level. This chapter summarizes in detail the method of experiments used to probe the cap-binding center of eIF4E, steady state and stopped-flow fluorescence, and microcalorimetry. The studies were performed with a wide class of synthetic, structurally modified cap analogs that resembles in some respect an application of site directed mutagenesis of the protein. The chapter presents a general recipe as to how to investigate protein-ligand interactions if the protein has no enzymatic activity and both the protein and the ligand absorb and emit UV/VIS radiation in the same spectral ranges.
Assuntos
Fator de Iniciação 4E em Eucariotos , Biossíntese de Proteínas , Análogos de Capuz de RNA , Capuzes de RNA , Animais , Calorimetria , Interpretação Estatística de Dados , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Camundongos , Estrutura Molecular , Ligação Proteica , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , Capuzes de RNA/metabolismo , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl2. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3' to 5' cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.
Assuntos
Endorribonucleases/metabolismo , Humanos , Hidrólise , Estrutura Molecular , Capuzes de RNA/síntese química , Capuzes de RNA/química , Capuzes de RNA/metabolismo , Especificidade por Substrato , TitulometriaRESUMO
All eukaryotic mRNAs contain a 5' terminal cap structure, which consists of 7-methylguanosine linked by a 5-5' triphosphate bridge to the first transcribed nucleoside (m7GpppN). Specific recognition of the cap by the eukaryotic initiation factor eIF4E plays a key role in regulation of translation initiation as a rate-limiting step. Using dynamic light scattering (DLS), the apo-form of murine eIF4E (33-217) was shown to aggregate. After addition of m7G7P, progressive deaggregation with the time of incubation in the presence of the cap analogue has been observed.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Capuzes de RNA , RNA Mensageiro/metabolismo , Animais , Escherichia coli/metabolismo , Cinética , Luz , Substâncias Macromoleculares , Camundongos , Modelos Químicos , Fatores de Iniciação de Peptídeos/química , Fosfatos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Prótons , Análogos de Capuz de RNA/química , Espalhamento de Radiação , Fatores de TempoRESUMO
A new methodology for synthesis of biologically important nucleoside tri- and tetraphosphates containing a bisphosphonate moiety instead of the terminal pyrophosphate bond is described. The series consists of tri- and tetraphosphate analogs of adenosine, guanosine and 7-methylguanosine (characteristic for mRNA cap). We have adopted a two-step procedure that allowed us to insert a methylene bridge into the phosphate chain. Nucleoside mono- or diphosphates were first activated (as imidazole derivatives) and then used in coupling reactions with organic salts of bisphosphonate. The resulting synthetic method enabled us to obtain the desired compounds with high yields and does not require any protective groups. This makes it very useful for the synthesis of labile compounds such as those containing the 7-methylguanosine ring. The structures of the synthesized compounds were confirmed by NMR spectroscopy. They were tested as potential substrates and inhibitors of several hydrolases.