The regulation of protein synthesis plays a major role in controlling gene expression and consequently cell growth, proliferation and differentiation. Rates of protein synthesis are altered in response to nutrient availability, hormones and growth factors and therefore are directly coupled with cell cycle progression. Our long-term goal is to understand the molecular mechanism and regulation of mammalian protein synthesis initiation. The recruitment of an mRNA to the ribosome requires the formation and maintenance of fundamental interactions between eukaryotic initiation factors (eIFs), the mRNA and the ribosome. The large eIF3 complex binds directly to the 40S ribosomal subunit, promoting mRNA recruitment through an insulin-regulated interaction with the eIF4G subunit of the cap-binding complex (eIF4E, eIF4G and eIF4A). The hypothesis is that specific interactions between the cap- binding complex, eIF3 and the 40S subunit are essential to recruit mRNA to the ribosome, promote helicase activity, and induce conformation rearrangements in the ribosome to initiate protein synthesis. We will test this hypothesis using biochemical and biophysical approaches to study the structural and kinetic basis of initiation factor functions in promoting important intermediates in the protein synthesis initiation pathway.
The Specific Aims are: I. Characterize the association of eIF4G with eIF3 and the 40S subunit. We will determine the consensus sequence in eIF4G that binds to eIF3. Important amino acids will be identified using the fluorescence polarization assay we have established together with site-directed mutagenesis of eIF4G. We will exploit these mutants to determine how this interaction regulates mRNA recruitment and protein synthesis in vivo. We will also reveal how a novel RNA binding region contributes to the stability of eIF4G with the eIF3-40S complex in vitro and in vivo. Subunits of eIF3 that interact with eIF4G will be identified using site-directed crosslinking. The role of identified eIF3 subunits will be confirmed by competition assays in vitro and in vivo. II. Elucidate how initiation factors promote structural changes in the 40S subunit enabling mRNA recruitment and AUG recognition. The mechanism by which the hepatitis c virus (HCV) internal ribosome entry site (IRES) functionally replaces the cap-binding complex in mRNA recruitment to the 40S subunit will be identified. Using a site-directed RNA cleavage assay we will determine the contribution that each initiation factor makes in promoting structural rearrangement of the 40S subunit during intermediates of the initiation pathway. This will provide a structural basis for initiation factor functions in mRNA recruitment. III. Determine how human ribosomes scan along the 5" untranslated region (5"UTR) to locate the AUG codon. We will establish a fluorescent kinetic RNA unwinding assay to determine how initiation factors contribute to the migration rate of the 40S subunit through secondary structure. The helicase activity of initiation factors and the 40S subunit during scanning will be visualized using a single-molecule assay, in collaboration with Steven Block (Stanford).

Public Health Relevance

PROJECT NARRATIVE Proteins catalyze most of the reactions on which life depends, so it is of no surprise that there are many links between the dysregulation of protein synthesis and cancer progression. An understanding of the mechanism and regulation of protein synthesis will therefore help us better understand this disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092927-04
Application #
8449267
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2010-04-05
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
4
Fiscal Year
2013
Total Cost
$271,878
Indirect Cost
$90,361
Name
University of California Davis
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
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Avanzino, Brian C; Fraser, Christopher S (2014) DEAD box unwinding caught in the act. Structure 22:931-2
Feoktistova, Kateryna; Tuvshintogs, Enkhee; Do, Angelie et al. (2013) Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Proc Natl Acad Sci U S A 110:13339-44
Villa, Nancy; Do, Angelie; Hershey, John W B et al. (2013) Human eukaryotic initiation factor 4G (eIF4G) protein binds to eIF3c, -d, and -e to promote mRNA recruitment to the ribosome. J Biol Chem 288:32932-40
Virgili, Genevieve; Frank, Filipp; Feoktistova, Kateryna et al. (2013) Structural analysis of the DAP5 MIF4G domain and its interaction with eIF4A. Structure 21:517-27
Sokabe, Masaaki; Fraser, Christopher S; Hershey, John W B (2012) The human translation initiation multi-factor complex promotes methionyl-tRNAi binding to the 40S ribosomal subunit. Nucleic Acids Res 40:905-13
Lee, Seung Bum; Park, Jong Hwan; Folk, John E et al. (2011) Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1). Biochem J 433:205-13
Ozes, Ali R; Feoktistova, Kateryna; Avanzino, Brian C et al. (2011) Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B. J Mol Biol 412:674-87
Andaya, Armann; Jia, Weitao; Sokabe, Masaaki et al. (2011) Phosphorylation of human eukaryotic initiation factor 2?: novel site identification and targeted PKC involvement. J Proteome Res 10:4613-23

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