Use of reticulocyte lysates for mechanistic studies of eukaryotic translation initiation.

This chapter describes how commercially available, nuclease-treated rabbit reticulocyte lysates can be used to study different types of translation initiation (cap-dependent initiation, reinitiation, internal ribosome entry site-mediated initiation) and the influence of different initiation factors on these translation mechanisms. Additionally, with the use of sucrose gradients, it is possible to use nuclease-treated reticulocyte lysates to monitor the formation of ribosomal complexes for their content of mRNA, initiator met-tRNA(i), and initiation factors. The advantage of using nuclease-treated lysates rather than purified initiation factors is that reactions occur at or near the in vivo rate in contrast to rates observed in reactions with purified components, which are generally 10- to 1000-fold lower.

Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A.

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The double eIF2A/eIF4E-ts mutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G(2)/M border.

p53 activation results in rapid dephosphorylation of the eIF4E-binding protein 4E-BP1, inhibition of ribosomal protein S6 kinase and inhibition of translation initiation.

p53 is an important regulator of cell cycle progression and apoptosis, and inactivation of p53 is associated with tumorigenesis. Although p53 exerts many of its effects through regulation of transcription, this protein is also found in association with ribosomes and several mRNAs have been identified that are translationally controlled in a p53-dependent manner. We have utilized murine erythroleukemic cells that express a temperature-sensitive p53 protein to determine whether p53 also functions at the level of translation.

S6 phosphorylation-independent pathways regulate translation of 5'-terminal oligopyrimidine tract-containing mRNAs in differentiating hematopoietic cells.

Synthesis of new ribosomes is an energy costly and thus highly regulated process. Ribosomal protein synthesis is controlled by regulating translation of the corresponding ribosomal protein (rp)mRNAs. In mammalian cells a 5'-terminal oligopyrimidine tract (TOP) is a conserved feature of these mRNAs that has been demonstrated to be essential for their translational regulation.