RESUMO
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
Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient protein synthesis in cap-dependent translation. The protein specifically binds the cap structure at the mRNA 5' terminus and facilitates the assembly of the mRNA with other initiation factors and the 40S ribosomal subunit. Phosphorylation of eIF4E is implicated in the regulation of the initiation step of translation. However, the molecular mechanism of this regulation still remains unclear. To address this problem, we have determined the binding affinities of eIF4E specifically mutated at position 209 or 159 for a series of novel mono- and dinucleotide cap analogues by a fluorometric time-synchronized titration method. A 1.5-3-fold reduction in the affinity of cap for the S209E mutant and a 1-2-fold increase in the affinity of cap for the S209K mutant, depending on the negative charge of phosphate chains, indicate that phosphorylation at Ser209 creates electrostatic repulsion between the protein and the negatively charged cap structure. The inhibition of the ability to bind cap analogues by the K159A mutant and its phosphorylated counterpart shows significant participation of Lys159 in the binding of the capped mRNA. Both structural modifications, phosphorylation and the replacement of lysine with alanine, result in an increase in the negative Gibbs free energy of association that is proportional to the length of the cap phosphate chain and additive, i.e., equal to the sum of the individual destabilizing changes of DeltaG degrees. The possible implication of these results for the mechanism of control of eIF4E by phosphorylation, especially for the "clamping model", is discussed.
Assuntos
Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Guanosina/análogos & derivados , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Alanina/genética , Animais , Ligação Competitiva/genética , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/genética , Guanosina/síntese química , Guanosina/metabolismo , Guanosina Pentafosfato/síntese química , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/síntese química , Guanosina Tetrafosfato/metabolismo , Humanos , Lisina/genética , Camundongos , Conformação de Ácido Nucleico , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica/genética , Capuzes de RNA/síntese química , Soluções , Eletricidade Estática , TermodinâmicaRESUMO
The bifunctional phosphorylating reagent O,O-bis[1-benzotriazolyl]phosphoromorpholidate was used to introduce a 5'-O-triphosphate and a 3'-O-diphosphate function in a partially protected riboguanosine. Pilot studies indicated that protection of the 2'-OH of ribonucleosides with the acid-labile tetrahydropyranyl group, instead of the more labile 4-methoxytetrahydropyran-4-yl group, was most satisfactory for the preparation of the Magic Spot II.
