Abstract

The long-term goal of the proposed research is a detailed structural and mechanistic understanding of the enormously complex macromolecular machine that controls translation initiation in eukaryotes. The proposed work builds on our structural and functional studies of mRNA selection by the cap-binding protein, eukaryotic initiation factor 4E or eIF4E. X-ray crystallography will be combined with chemical methods to study the structures and mechanisms of action of an important subset of the remaining translation initiation factors [eIF4G, eIF4A, eIF2, IF2, eIF3, eIF1A, eIF2B, eIF5, the poly (A)-binding protein (PABP), and a PABP-interacting protein]. These translation factors participate in the following biochemical steps: (a) resolving secondary structural features in the 5' untranslated region of the mRNA, (b) delivering the methionyl initiator tRNA to the 40S subunit, (d) recycling the G protein that loads the methionyl initiator tRNA onto the 40S subunit, (d) recruitment of the 40S subunit, the methionyl initiator tRNA and various accessory factors to the 5' untranslated region of cellular mRNAs bearing 5' 7-methyl-G caps, (e) recruitment of the same components to the 5' untranslated region of an uncapped viral RNA, (f) 60S ribosomal subunit joining, and (g) synergy of transcription initiation via mRNA circularization by the PABP.
The specific aims of the research are as follows:
Specific Aim 1. Determine X-ray structures of the RNA helicase eIF4A, and its complexes with ADP, a non-hydrolyzable ATP analog, and single-stranded RNA.
Specific Aim 2. Determine X-ray structures of eIF2, and its complexes with GDP, a non-hydrolyzable GTP analog, and methionyl initiator tRNA.
Specific Aim 3. Determine the X-ray structure of eukaryotic IF2.
Specific Aim 4. Determine X-ray structures of a C-terminal fragment of eIF4G, and its complex with a picornavirus internal ribosome entry site.
Specific Aim 5. Determine X-ray structures of eIF5 and eIF2Bepsilon, and their complexes with eIF2beta.
Specific Aim 6. Determine X-ray structures of PABP recognizing poly (A) RNA, and various binary and ternary complexes with a PABP-interacting protein and eIF4G.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061262-05
Application #
6636448
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
2000-03-01
Project End
2005-05-31
Budget Start
2003-03-01
Budget End
2005-05-31
Support Year
5
Fiscal Year
2003
Total Cost
$418,941
Indirect Cost

  Institution

Name
Sgx Pharmaceuticals, Inc.
Department
Type
DUNS #
086919938
City
San Diego
State
CA
Country
United States
Zip Code
92121

  Publications

Robert, Francis; Williams, Chris; Yan, Yifei et al. (2009) Blocking UV-induced eIF2alpha phosphorylation with small molecule inhibitors of GCN2. Chem Biol Drug Des 74:57-67
Bellsolell, Lluis; Cho-Park, Park F; Poulin, Francis et al. (2006) Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1. Structure 14:913-23
Li, Keqin; Zhao, Kehao; Ossareh-Nazari, Batool et al. (2005) Structural basis for interaction between the Ubp3 deubiquitinating enzyme and its Bre5 cofactor. J Biol Chem 280:29176-85
Padyana, Anil K; Qiu, Hongfang; Roll-Mecak, Antonina et al. (2005) Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2alpha protein kinase GCN2. J Biol Chem 280:29289-99
Niessing, Dierk; Huttelmaier, Stefan; Zenklusen, Daniel et al. (2004) She2p is a novel RNA binding protein with a basic helical hairpin motif. Cell 119:491-502
Roll-Mecak, Antonina; Alone, Pankaj; Cao, Chune et al. (2004) X-ray structure of translation initiation factor eIF2gamma: implications for tRNA and eIF2alpha binding. J Biol Chem 279:10634-42
Shin, Byung-Sik; Maag, David; Roll-Mecak, Antonina et al. (2002) Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity. Cell 111:1015-25
Groft, Caroline M; Burley, Stephen K (2002) Recognition of eIF4G by rotavirus NSP3 reveals a basis for mRNA circularization. Mol Cell 9:1273-83
Marcotrigiano, J; Lomakin, I B; Sonenberg, N et al. (2001) A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery. Mol Cell 7:193-203
Roll-Mecak, A; Shin, B S; Dever, T E et al. (2001) Engaging the ribosome: universal IFs of translation. Trends Biochem Sci 26:705-9

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