Mechanism of Eukaryotic Translation Termination

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DESCRIPTION (provided by applicant): In eukaryotic organisms, eRF1 recognizes the three translation termination codons and mediates the release of nascent polypeptide chains. The accessory factor eRF3 assists the termination process in a GTP-dependent manner. Over the last few years, our understanding of how eRF1 recognizes stop codons and facilitates polypeptide chain release has increased, but much work remains to be done. In contrast, little has been done to understand how GTP hydrolysis by eRF3 assists in the termination process. Unlike prokaryotes, eRF3 is an essential gene in yeast. Furthermore, eRF3 has been shown to interact not only with eRF1, but also with other proteins involved in various aspects of mRNA function and metabolism, including poly(A)-binding protein and Upflp, Upf2p, and Upf3p. This suggests that eRF3 may act not only in translation termination, but also couple the termination process to other related cellular functions such as mRNA stability, NMD, and possibly translation initiation. To better understand how eRF1 and eRF3 function in translation termination, we propose the following Specific Aims: Specific Aim 1): Determine the function of eRF3 in translation termination. We will couple a mutational analysis of eRF3 to in vitro and in vivo functional assays to determine the role of eRF3 in this process. Specific Aim 2): Determine how eRF1 and the ribosome influence eRF3 function in translation termination. We will use functional assays to determine how these factors stimulate the GTPase activity of eRF3. Specific Aim 3): Determine whether phosphorylation and/or methylation of eRF1 modulates its function during translation termination. We will use mass spectrometry to characterize the post-translational modification of eRF1. We will then examine how phosphorylation influences eRF1 function, and determine whether the highly conserved GGQ motif is methylated in eukaryotes. Specific Aim 4): Determine how two new factors, Pst21p and Slhlp, influence the efficiency of translation termination. We will examine whether these factors interact directly with eRF1, eRF3, or Upflp, and examine functional motifs in these proteins that may provide clues to their cellular function.