RESUMO
The abundance of noncanonical mechanisms of eukaryotic initiation of translation indicates their involvement in the regulation of protein synthesis during key events in a cell life. One of the well-known examples of a noncanonical cap-independent process is the initiation of translation of mRNA with the 5'-untranslated (leader) region of the messenger encoding for the photoprotein obelin (the obelin leader). In the present work, mRNA with the obelin leader was modified by adding 45 deoxycytidyl nucleotides and a fluorescent label to its 5'end. Formation of the 48S ribosomal initiation complexes at the start codon of the modified mRNA was studied using primer extension inhibition (toeprinting). In contrast to mRNA with the intact obelin leader, translation initiation of which strictly requires the eIF4F factor, initiation on the modified mRNA can take place in the absence of this factor, although with less efficiency. The finding thus indicates the unknown function of the eIF4F factor in the first step(s) of mRNA recognition by ribosomal subunits.
Assuntos
Regiões 5' não Traduzidas , Fator de Iniciação 4F em Eucariotos , Fatores de Iniciação de Peptídeos , Biossíntese de Proteínas , Códon de Iniciação , Eucariotos , Fator de Iniciação 4F em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Ribossomos/metabolismoRESUMO
The formation of ribosomal 48S initiation complexes at the start codon of the mRNA leader sequence that encodes obelin has been studied using the method of primer extension inhibition (toeprinting). Experiments have been performed in a system composed of purified individual components required to initiate translation. The influence of the dominant negative mutant of factor eIF4A (R362Q) on translation initiation has been studied. It has been shown that the presence of the mutant in reaction mixture has no effect on efficiency of formation of the 48S complexes at start codon of the template studied.
Assuntos
Códon de Iniciação/genética , Fator de Iniciação 4F em Eucariotos/genética , Proteínas Luminescentes/genética , RNA Mensageiro/genética , Ribossomos/genética , AnimaisRESUMO
The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex.
Assuntos
Fator de Iniciação 4A em Eucariotos/química , Fatores de Iniciação em Eucariotos/química , Iniciação Traducional da Cadeia Peptídica , Ribossomos/química , Animais , Sistema Livre de Células , Fator de Iniciação 4A em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Ribossomos/metabolismoRESUMO
The objective of the present work was to determine whether it is possible to use a nonhydrolyzable analog of ATP (AMP-PNP) as an inhibitor of ATP-dependent scanning of the leader sequence of eukaryotic mRNA in translation initiation-. The formation of ribosomal 48S initiation complexes at the start codon of the capped mRNA leader sequence of rabbit ß-globin mRNA was studied. The study was carried out in a system composed of individual components of translation initiation. The dependences of the efficiency of formation of 48S initiation complexes on ATP concentration and incubation time were obtained in the absence and presence of AMP-PNP. It was found that AMP-PNP did not affect the efficiency of formation of 48S initiation complexes in all cases under study. We conclude that the uncleavable analog of ATP, AMP-PNP, is not an inhibitor of translation initiation in eukaryotes.
