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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068854-04
Application #
7067093
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
2003-06-01
Project End
2007-11-30
Budget Start
2006-06-01
Budget End
2007-11-30
Support Year
4
Fiscal Year
2006
Total Cost
$273,982
Indirect Cost
Name
University of Alabama Birmingham
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Kelly, Shane P; Bedwell, David M (2015) Both the autophagy and proteasomal pathways facilitate the Ubp3p-dependent depletion of a subset of translation and RNA turnover factors during nitrogen starvation in Saccharomyces cerevisiae. RNA 21:898-910
Conard, Sara E; Buckley, Jessica; Dang, Mai et al. (2012) Identification of eRF1 residues that play critical and complementary roles in stop codon recognition. RNA 18:1210-21
Keeling, Kim M; Bedwell, David M (2011) Suppression of nonsense mutations as a therapeutic approach to treat genetic diseases. Wiley Interdiscip Rev RNA 2:837-52
Vallabhaneni, Haritha; Fan-Minogue, Hua; Bedwell, David M et al. (2009) Connection between stop codon reassignment and frequent use of shifty stop frameshifting. RNA 15:889-97
Fan-Minogue, Hua; Du, Ming; Pisarev, Andrey V et al. (2008) Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination. Mol Cell 30:599-609
Fan-Minogue, Hua; Bedwell, David M (2008) Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity. RNA 14:148-57
Keeling, Kim M; Salas-Marco, Joe; Osherovich, Lev Z et al. (2006) Tpa1p is part of an mRNP complex that influences translation termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae. Mol Cell Biol 26:5237-48
Salas-Marco, Joe; Fan-Minogue, Hua; Kallmeyer, Adam K et al. (2006) Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes. Mol Cell Biol 26:438-47
Kallmeyer, Adam K; Keeling, Kim M; Bedwell, David M (2006) Eukaryotic release factor 1 phosphorylation by CK2 protein kinase is dynamic but has little effect on the efficiency of translation termination in Saccharomyces cerevisiae. Eukaryot Cell 5:1378-87
Salas-Marco, Joe; Bedwell, David M (2005) Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough. J Mol Biol 348:801-15

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