Eukaryotes translation initiation

Gomez, E., and Pavitt, G. D. (2000). Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation. Mol. Cell Biol. 20, 3965—3976. 

Singh, C. R., Curtis, C., Yamamoto, Y., Hall, N. S., Kruse, D. S., He, H., Hannig, E. M., and Asano, K. (2005). Eukaryotic translation initiation factor 5 is critical for integrity of the scanning preinitiation complex and accurate control of GCN4 translation. Mol. Cell. Biol. 25, 5480-5491. 

Valasek, L., Nielsen, K. H., Zhang, F., Fekete, C. A., and Hinnebusch, A. G. (2004). Interactions of Eukaryotic Translation Initiation Factor 3 (eIF3) Subunit NIPl/c with elFl and eIF5 promote preinitiation complex assembly and regulate start codon selection. Mol. Cell. Biol. 24, 9437-9455. 

Genes of interest can be tagged at either the N or C terminal end. The decision to tag a protein at either the N or the C terminal depends upon the properties of the protein of interest. In our case, all the eukaryotic translation initiation factors were tagged C terminally to allow the endogenous promoter to influence the expression of the tagged protein. 

In studying the localization of eukaryotic translation initiation factors, we made use of the FR AP technique to determine whether the localized regions of eIF2/eIF2B represented dynamic centers of these proteins (Campbell et ah, 2005). 

McEwen, E., Kedersha, N., Song, B., Scheuner, D., Gilks, N., Han, A., Chen, J. J., Anderson, P., and Kaufman, R. J. (2005). Heme-regulated inhibitor (HRI) kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 (eIF2) inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure. J. Biol. Chem. 280, 16925—16933. 

Kahvejian, A., Svitkin, Y. V., Sukarieh, R., M Boutchou, M. N., and Sonenberg, N. (2005). Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms. Genes Dev. 19, 104—113. 

Smirnova, J. B., Selley, J. N., Sanchez-Cabo, F., Carroll, K., Eddy, A. A., McCarthy, J. E., Hubbard, S. J., Pavitt, G. D., Grant, C. M., and Ashe, M. P. (2005). Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways. Mol. Cell Biol. 25, 9340-9349. 

Identifying Small Molecule Inhibitors of Eukaryotic Translation Initiation... 

In eukaryotes, translation initiation is rate-limiting with much regulation exerted at the ribosome recruitment and ternary complex (elF2 GTP Met-tRNAjMet) formation steps. Although small molecule inhibitors have been extremely useful for chemically dissecting translation, there is a dearth of compounds available to study the initiation phase in vitro and in vivo. In this chapter, we describe reverse and forward chemical genetic screens developed to identify new inhibitors of translation. The ability to manipulate cell extracts biochemically, and to compare the activity of small molecules on translation of mRNA templates that differ in their factor requirements for ribosome recruitment, facilitates identification of the relevant target. 

Figure 13.1 Schematic diagram of eukaryotic translation initiation. The sites of action of small molecule inhibitors are shown with dashed lines. Kinases that affect the phosphorylation of 4E-BP and eIF2a, and exert effects on ribosome recruitment and ternary complex formation, respectively, are shown in a black box. See text for details.

A novel functional human eukaryotic translation initiation factor 4G. Mol. Cell Biol. 18, 334-342. 

Imataka, H., and Sonenberg, N. (1997). Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A. Mol. Cell Biol. 17, 6940-6947. 

Polunovsky, V. A., Rosenwald, I. B., Tan, A. T., White, J., Chiang, L., Sonenberg, N., and Bitterman, P. B. (1996). Translational control of programmed cell death Eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc. Mol. Cell Biol. 16, 6573-6581. 

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnkl to phosphorylate eIF4E. EMBO J. 18, 270-279. 

Sizova, D. V., Kolupaeva, V. G., Pestova, T. V., Shatsky, I. N., and Hellen, C. U. (1998). Specific interaction of eukaryotic translation initiation factor 3 with the 5 nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J. Virol. 72, 4775-4782. 

Abbreviations aa-tRNA Amino-acyl tRNA eLF Eukaryotic translation initiation factor IF Prokaryotic translation initiation factor eEF Eukaryotic translation elongation factor EF Prokaryotic translation elongation factor eRF Eukaryotic translation termination factor (release factor) RF Prokaryotic translation release factor RRF Ribosome recycling factor Rps Protein of the prokaryotic small ribosomal subunit Rpl Protein of the eukaryotic large ribosomal subunit S Protein of the prokaryotic small ribosomal subunit L Protein of the prokaryotic large ribosomal subunit PTC Peptidyl transferase center RNC Ribosome-nascent chain-mRNA complex ram Ribosomal ambiguity mutation RAC Ribosome-associated complex NMD Nonsense-mediated mRNA decay... 

Wang W, Czaplinski K, Rao Y, Peltz SW (2001) The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts. EMBO J 20 880-890 Wells SE, Hillner PE, Vale RD, Sachs AB (1998) Circularization of mRNA by eukaryotic translation initiation factors. Mol Cell 2 135-140... 

Eukaryotic translation initiation is far more complicated than the prokaryotic system described earlier. To begin the process, two initiation factor... 

A. J. Waskiewicz, et al. Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnkl in vivo. Mol Cell Biol, 19, 1871-1880, 1999. 

Figure 29.33. Eukaryotic Translation Initiation. In eukaryotes, translation initiation starts with the assembly of a complex on the 5 cap that includes the 40S subunit and Met-tRNAj. Driven by ATP hydrolysis, this complex scans the mRNA until the first AUG is reached. The 60S subunit is then added to form the SOS initiation complex.

S. Das, R. Ghosh, and U. Maitra. 2001. Eukaryotic translation initiation factor 5 functions as a GTPase-activating protein J. Biol. Chem. 276 6720-6726. (PubMed)... 

Eukaryotes utilize many more initiation factors than do prokaryotes, and their interplay is much more intricate. The prefix elF denotes a eukaryotic initiation factor. For example, eIF-4E is a protein that binds directly to the 7-inethylguanosine cap (p. 846), whereas eIF-2, in association with GTP, delivers the met-tRNA to the ribosome. The difference in initiation mechanism between prokaryotes and eukaryotes is, in part, a conseciuence of the ence in RNA processing. The 5 end of mRNA is readily available to ribosomes immediately after transcription in prokaryotes. In contrast, pre-mRNA must be processed and transported to the cytoplasm in eukaryotes before translation is initialed. The 5 cap provides an easily recognizable starting point. In addition, the complexity of eukaryotic translation initiation provides another mechan ism for regulation of gene expression that we shall explore further in Chapter 31. 

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