Our laboratory studies how protein synthesis is initiated and regulated in the yeast Saccharomyces cerevisiae. Messenger RNA (mRNA) is bound at its capped 5'-end and poly(A)-tailed 3'-end by the eIF4E and Pab1 proteins, respectively. These interact with a scaffolding protein, eIF4G, to circularize the mRNA and to probably provide optimal binding sites for translation initiation factors. Together these factors mediate the process by which the small ribosomal subunit binds to the 5'-end of the mRNA and then searches for the initiator methionine codon. We are discovering the identity of some of these factors, how many of them function, and how they are regulated.
The yeast eIF4G protein contains binding surfaces for eIF4E, Pab1p, and the RNA-stimulated ATPase eIF4A. The regulation of eIF4E binding to eIF4G is being studied through the identification and characterization of other proteins that either stimulate or inhibit it. The binding of Pab1p to eIF4G is probably only one of its contacts within the translational apparatus. We are pursuing our findings that Pab1p can interact directly with ribosomal subunits, and that in vitro this interaction requires the activity of other proteins normally bound to the ribosome. The function of eIF4A in translation is also modulated by factors other than eIF4G, and we are using multiple methods to identify and characterize these modulators.
The appropriate, rapid response of cells to changes in their nutritional status prevents cell death under certain conditions. We have recently shown that glucose deprivation leads to a rapid shut off of translation in yeast, and are now trying to understand the molecular mechanisms underlying this response. We are also developing novel reagents to measure gene expression in yeast. These will allow for the rapid purification of mRNA bound to various mRNA binding proteins or the ribosome, and for the quantitative measure of gene expression in living cells.
Circularization of RNA by the eIF4E, eIF4G, and Pab1 proteins. Complexes were assembled in vitro and then visualized in liquid by atomic force microscopy. The large white particles connecting the two ends of the RNA contain each of the three proteins. See (Wells et al., 1998) for details.
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