Dysregulated translaion initiation causes malignant transformation or other chronic diseases by different mechanisms. Therefore, studies on the mechanism of translation initiation help to enhance human health and well being. The long-term goal of this proposal is to understand the complex pathway leading to the accurate initiation of translation. In translation initiation, eukaryotic initiation factor 1 (elF1), elF5, and all three subunits of elF2 are implicated in the stringent AUG selection for yeast mRNAs. The C-terminal domain (CTD) of elF5 binds elF1 and bridges interaction between elF2 and a five-subunit factor elF3, mediating fomration of the multifactor complex (MFC) which also contains the methionyl initiator tRNA. When bound to the ribosome, the MFC interactions rearrange, allowing elF5-CTD to interact with the elF4G subunit of elF4F complex bound to S'-capped mRNA and promoting formation of ribosomal preinitiation complex. Under the previous aims in this GM67841 proposal, evidence was provided that the accuracy of translation initiation depends on the function of the MFC, but how this complex assembles and functions is still unclear. In particular, it is very important to correlate the functions of the MFC and preinitiation complexes to the structure of individual elFs, which has started to unveil. In collaboration with Dr. Gerhard Wagner at Harvard Medical School, the Asano group has solved the structure of yeast elF1 and identified its interfaces to the NTDs of elF2? and elFSc and the CTDs of elF4G and elF5. Genetic and biochemical studies showed how these interactions may rearrange during preinitiation complex assembly and function. In this renewal proposal, the first aim is to solve the structure of elF5-CTD in collaboration with the Wagner group, and use this information to direct its well-designed mutagenesis studies.
The second aim i s to reconstitute full MFC from purified constituents, and use it to understand the mechanism of its assembly activation and hypothetical control by elF5 phosphorylation. Finally, the third aim is to study the critical partners of the MFC in the preinitiation complex, elF4G-CTD and the ribosomal RNA. The data obtained from the previous aims will be used to predict elF5-CTD-binding face of elF4G-CTD and perform site-directed mutagenesis studies on the latter. >30 Ts- mutations mapping in rRNA obtained under an aim in the previous proposal will be used together with new mutations to be introduced at the predicted 40S subunit interface to mRNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064781-08
Application #
7540476
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
2002-01-01
Project End
2010-12-31
Budget Start
2009-01-01
Budget End
2009-12-31
Support Year
8
Fiscal Year
2009
Total Cost
$277,400
Indirect Cost
Name
Kansas State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
929773554
City
Manhattan
State
KS
Country
United States
Zip Code
66506
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Hiraishi, Hiroyuki; Shin, Byung-Sik; Udagawa, Tsuyoshi et al. (2013) Interaction between 25S rRNA A loop and eukaryotic translation initiation factor 5B promotes subunit joining and ensures stringent AUG selection. Mol Cell Biol 33:3540-8
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Luna, Rafael E; Arthanari, Haribabu; Hiraishi, Hiroyuki et al. (2013) The interaction between eukaryotic initiation factor 1A and eIF5 retains eIF1 within scanning preinitiation complexes. Biochemistry 52:9510-8
Luna, Rafael E; Arthanari, Haribabu; Hiraishi, Hiroyuki et al. (2012) The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2?. Cell Rep 1:689-702
Singh, Chingakham Ranjit; Watanabe, Ryosuke; Chowdhury, Wasimul et al. (2012) Sequential eukaryotic translation initiation factor 5 (eIF5) binding to the charged disordered segments of eIF4G and eIF2ýý stabilizes the 48S preinitiation complex and promotes its shift to the initiation mode. Mol Cell Biol 32:3978-89

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