Translation eukaryotes

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. 

An Approach to Studying the Localization and Dynamics of Eukaryotic Translation Factors in Live Yeast Cells... 

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. 

Waskiewicz, A. J., Johnson, J. C., Penn, B., Mahalingam, M., Kimball, S. R., and Cooper, J. A. (1999). Phosphorylation of the cap-binding protein eukaryotic translation factor 4E by protein kinase Mnkl in vivo. Mol. Cell. Biol. 19, 1871—1880. 

Arava, Y., Boas, F. E., Brown, P. O., and Herschlag, D. (2005). Dissecting eukaryotic translation and its control by ribosome density mapping. Nucleic Acids Res. 33, 2421-2432. 

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. 

This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and amino-acylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model universal mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a ... 

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.

Bordeleau, M. E., Matthews, J., Wojnar, J. M., Lindqvist, L., Novae, O., Jankowsky, E., Sonenberg, N., Northcote, P., Teesdale-Spitde, P., and Pelletier, J. (2005). Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation. Proc. Natl. Acad. Sci. USA 102, 10460—10465. 

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. 

There is no doubt that plants represent one of the most productive and yet inexpensive sources of biomass. The absence of contaminating animal pathogens, the eukaryotic translational machinery and the ease of plant virus manipulation make plants... 

Some well-known inhibitors of prokaryotic translation include streptomycin, erythromycin, tetracydine, and chloramphenicol. Inhibitors of eukaryotic translation include cycloheximide and Diphtheria and Pseudomonas toxins. 

Puromycin inhibits both prokaryotic and eukaryotic translation by binding to the A site. Peptidyl transferase attaches the peptide to puromycin, and the peptide with puromycin attached at the C-terminus is released, prematurely terminating chain growth. 

Answer C. eIF-2 designates a protein factor of the initiation phase in eukaryotic translation. The only event listed that would occur during this phase is placement of initiator tRNA in the P-site. 

Both the cap and the poly-A tail play a vital part in initiating eukaryotic translation (see p. 250). They help position the ribosome correctly on the mRNA near to the starting codon. The protection which the additional nucleotides provide against premature enzymatic degradation appears to be of lesser importance. 

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... 

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