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).
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.
|Avanzino, Brian C; Jue, Helen; Miller, Clare M et al. (2018) Molecular mechanism of poliovirus Sabin vaccine strain attenuation. J Biol Chem 293:15471-15482|
|Sokabe, Masaaki; Fraser, Christopher S (2017) A helicase-independent activity of eIF4A in promoting mRNA recruitment to the human ribosome. Proc Natl Acad Sci U S A 114:6304-6309|
|Avanzino, Brian C; Fuchs, Gabriele; Fraser, Christopher S (2017) Cellular cap-binding protein, eIF4E, promotes picornavirus genome restructuring and translation. Proc Natl Acad Sci U S A 114:9611-9616|
|Rossi, Danuza; Barbosa, Natalia M; Galvão, Fabio C et al. (2016) Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome. PLoS One 11:e0154205|
|Barbosa, Natália M; Boldrin, Paulo E G; Rossi, Danuza et al. (2016) Mapping surface residues of eIF5A that are important for binding to the ribosome using alanine scanning mutagenesis. Amino Acids 48:2363-74|
|Fraser, Christopher S (2015) Quantitative studies of mRNA recruitment to the eukaryotic ribosome. Biochimie 114:58-71|
|Sidrauski, Carmela; Tsai, Jordan C; Kampmann, Martin et al. (2015) Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. Elife 4:e07314|
|García-García, Cuauhtémoc; Frieda, Kirsten L; Feoktistova, Kateryna et al. (2015) RNA BIOCHEMISTRY. Factor-dependent processivity in human eIF4A DEAD-box helicase. Science 348:1486-8|
|Sokabe, Masaaki; Fraser, Christopher S (2014) Human eukaryotic initiation factor 2 (eIF2)-GTP-Met-tRNAi ternary complex and eIF3 stabilize the 43 S preinitiation complex. J Biol Chem 289:31827-36|
|Avanzino, Brian C; Fraser, Christopher S (2014) DEAD box unwinding caught in the act. Structure 22:931-2|
Showing the most recent 10 out of 21 publications