Protein synthesis is one of the multiple levels at which gene expression is regulated. The rate-limiting steps of protein synthesis occur during initiation. The binding of the ribosome to the mRNA in translation initiation is catalyzed by the proteins of the eukaryotic initiation factor 4 (eIF4) group. In mammals, the mRNA cap-binding protein eIF4E is present in limiting levels and is regulated by several mechanisms. This thesis examines the regulation of eIF4E during the development of the genetically tractable organism, Drosophila melanogaster. A Drosophila eIF4E gene was cloned, its position was mapped cytologically, and this gene was shown to encode two cap-binding protein isoforms via alternative splicing. Antisera specific to the eIF4E isoforms were raised and purified to characterize the expression of eIF4E during development. Several mutant alleles of eIF4E were identified and demonstrate that this gene is essential for the viability of Drosophila. Furthermore, eIF4E mutants arrest in growth during early larval stages. The lethality and growth defects of eIF4E mutant alleles were rescued by a transgene containing a wild-type copy of eIF4E expressed under the control of its endogenous promoter. Ser251 of Drosophila eIF4E is in a sequence context identical to site on which eIF4E is phosphorylated in response to extracellular stimuli in other organisms. To examine the biological significance of the phosphorylation of eIF4E, transgenic flies were generated in which Ser251 was mutated. We show that eIF4E from Ser251 mutant lines cannot incorporate labeled orthophosphate. Interestingly, flies in which the only source of eIF4E is non-phosphorylatable are semi-lethal and escapers are small in size. These results are evidence that Ser251 of eIF4E is required for the normal growth of a multicellular organism.

Thesis (Ph.D.) OGI School of Science & Engineering at OHSU, March 2008. / Includes bibliographical references (leaves 185 - 191).

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) responsible for the inhibition of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining, and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α, we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

No description available.

No description available.

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2004. / Title from second page of PDF document. Includes bibliographical references (p. 139-145).

Purpose: To determine the effect of four postexercise beverages, differing in macronutrient content, on metabolic response to an acute resistance exercise bout. Methods: Forty male subjects performed five sets of eight repetitions at 80% 1RM for leg press and leg extension, and then consumed one of four postexercise beverages (Placebo, PL: a carbohydrate-electrolyte beverage, CE; or one of two milk-based beverages, MILK 1: 1% chocolate milk; MILK 2: a high protein milk beverage). Indicators of muscle protein synthesis (MPS) were assessed before and 1-hr after consuming a postexercise beverage. Muscle protein degradation (MPD) was examined the day before and the day of exercise. Results: No significant differences were found among groups in MPS. The resistance exercise bout increased the amount of eIF4E-eIF4G by 4.5% 1-hr postexercise (p<0.05) without affecting the amount of eIF4E-4E-BP1. One hour after beverage consumption, serum total amino acid concentration increased for MILK 1 (p=0.003) and MILK 2 (p<0.001) but decreased for CE (p=0.028) and PL (p=0.276). Consumption of MILK 1, MILK 2, and CE significantly increased circulating levels of serum insulin (p<0.001). Serum growth hormone increased 3-fold as a result of the exercise bout but fell to baseline for all groups by 60 min (p<0.001). Conclusion: The resistance exercise bout was anabolic as shown by the increase in the active eIF4E-eIF4G complex and serum growth hormone. Consumption of MILK 2 led to the most optimal environment for muscle anabolism; however, none of the experimental beverages influenced the measured indicators of muscle protein translation 1-hr after ingestion. / Master of Science

<p>Initiation of protein synthesis in <i>E.coli </i>involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation. </p><p>We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNA^fMet. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes.</p><p>The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNA^fMet (initiator tRNA), Met-tRNA^fMet(non-formylated initiator tRNA) and Phe-tRNA^Phe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNA^fMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes.</p><p>Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNA^fMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNA^fMet by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis. </p>

Initiation of protein synthesis in E.coli involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation. We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNAfMet. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes. The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNAfMet (initiator tRNA), Met-tRNAfMet (non-formylated initiator tRNA) and Phe-tRNAPhe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNAfMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes. Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNAfMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNAfMet by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T16:16:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-LarissaNascimento.pdf: 5009861 bytes, checksum: e41e4e6a4cc83034688c263b000766c1 (MD5) / Made available in DSpace on 2017-04-10T16:16:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-LarissaNascimento.pdf: 5009861 bytes, checksum: e41e4e6a4cc83034688c263b000766c1 (MD5) Previous issue date: 2012-03-13 / O gênero Leishmania compreende 30 espécies de protozoários flagelados pertencentes a famíla Trypanosomatidae, donde 20 são patogênicas ao homem. Esses organismos apresentam ciclos de vida complexos e peculiaridades moleculares frente à maioria dos eucariontes, como a ausência de regulação transcricional. Desse modo, a regulação da expressão gênica nesses parasitas é efetuada em etapas pós-transcricionais, dentre essas a mais importante é o processo de iniciação da tradução dos mRNAs, onde diferentes fatores denominados eIFs (eukariotic initiation factors) estão envolvidos. Dentre esses fatores se destaca o complexo eIF4F com função de promover o reconhecimento e ligação de RNAs maduros aos ribossomos. Tal complexo é composto de três sub-unidades: eIF4A (RNA helicase); eIF4E (proteína de ligação ao cap); e eIF4G (proteína multidomínio estruturadora do complexo eIF4F). Em tripanossomatídeos se sabe da existência de cinco homólogos da sub-unidade eIF4G distintos (EIF4G1 ao G5), contudo, pouco se sabe sobre a ocorrência e funções celulares desses homólogos. Portanto o objetio do presente trabalho foi avaliar a expressão dos diferentes homólogos do eIF4G durante o ciclo de vida de Leishmania amazonensis, caracterizando as possíveis modificações pós-traducionais por fosforilação que possam estar agindo sobre tais fatores, uma vez que em eucariotos superiores mecanismos de regulação global da tradução por fosforilação dos eIFs via MAP quinases já são conhecidos. Para tal, foram realizadas culturas de L. amazonensis nas formas promastigota e amastigota-axênicas, e os extratos protéicos provenientes de diferentes fases do crescimento foram analisados através de Western blot. Foi observado que os homólogos de eIF4G estão presentes durante todo o ciclo de vida de L. amazonensis. Podendo ser observado que os EIF4G1, G4 e G5 apresentaram mais de uma isoforma proteica sugestiva de possíveis modificações pós-traducionais desses homólogos. Em conseguinte, a expressão de EIF4G3 e EIF4G4 foi analisada em condições especiais de cultivo na presença de seis inibidores diferentes, contudo nenhuma dessas condições alterou a expressão desses fatores, revelando que essas proteínas são bastante estáveis e possuem tempo de meia-vida prolongado. Posteriormente, o mapeamento in silico de sítios de fosforilação por MAP quinases nos EIF4Gs de Leishmania spp. demonstra a existência de sítios de fosforilação específicos em todos os homólogos E a purificação de fosfoproteínas confirma a existência de mecanismos de fosforilação agindo nos EIF4G3 e EIF4G4. Esses resultados auxiliam no esclarecimento dos mecanismos moleculares, até então obscuros, envolvidos na regulação da expressão gênica pós-transcricional característica desses organismos. / The genus Leishmania comprises 30 species of flagellated protozoa from the family Tripanosomatidae, of which 20 are pathogenic to humans. These organisms have complex life cycles and molecular particularities not observed in most eukaryotes, like absence of transcriptional regulation. Thus, the regulation of gene expression occurs in post-transcriptional steps, with the initiation of mRNA translation representing the most important event. Different factors called eIFs (eukariotic initiation factors) are involved, with emphasis in eIF4Fcomplex, which promotes mRNA recognition and its ribosome interaction. This eIF4F complex consists of three subunits: eIF4A (RNA helicase), eIF4E (cap binding protein) and eIF4G (scaffold protein, for eIF4F complex maintenance). In trypanosomatids, five eIF4G homologous (EIF4G1 to G5) was described, however, little is known about the specific cellular functions of these homologous. In this manner, we evaluated the expression and characterized post-translational modifications of the eIF4G homologous during the Leishmania amazonensis life cycle, especially phosphorylation, considering the translational regulation by phosphorylation of eIF ́s via MAP kinases observed in higher eukaryote. For this reason, cultures were performed with the L. amazonensis promastigote and amastigote axenic forms, and the protein extract, collected from different growth phases, were analyzed by Western blotting. These results demonstrated the detection of all eIF4G homologues during the life cycle of L. amazonensis, while more than one protein isoform were observed for the EIF4G1, G4 and G5 homologues, suggesting possible post-translational modifications. Posteriorly, the EIF4G3 and EIF4G4 gene expression were investigated under differential growth conditions using six distinct inhibitors, however no change was observed in the expression pattern, which suggests that these proteins are quite stable and have long half-life extending. Finally, In silico analysis shows specifics MAP kinase-dependent phosphorylation sites presents in all eIFG homologues and further analysis with EIF4G3 and EIF4G4 confirmed the existence of phosphorylation mechanisms acting on these factors.These present data help to clarify the molecular mechanisms involved in post-transcriptional regulation of gene expression characteristic of these organisms.