Control of translation initiation is important in a wide range of normal biological processes, from development to learning and memory to cell death. Improper execution and control of translation initiation are involved in the etiology of a variety of human diseases, including viral infection, neurodegeneration and cancer. In order to understand how translation initiation is controlled, and how this control can break down, we must first understand the underlying molecular mechanics of the normal process. The experiments proposed in this application will deepen our understanding of the molecular basis of translation initiation in eukaryotes. The work will employ a fully reconstituted S. cerevisiae translation initiation system and will involve close collaboration with the laboratory of Alan Hinnebusch at the NIH/NICHD. The studies proposed will address two major gaps in our understanding of eukaryotic translation initiation.
The first aim focuses on the mechanism through which the start codon in an mRNA is recognized by the ribosomal pre-initiation complex. Recognition of the start codon is one of the most important readings of the genetic code because it sets the frame for decoding of the message. Improper start site selection results in production of a miscoded, potentially toxic, protein. The proposed studies will elucidate the pathways of information transfer within the pre-initiation complex, from initial formation of three base pairs between the start codon and initiator tRNA anticodon to the downstream irreversible events that commit the complex to finishing initiation at the selected point on the message. Our studies will focus on the roles that the initiator tRNA itself and the initiation factor eIF5 play in this process. A significant amount of data indicates that these two components are missing links in our understanding of the molecular basis of start codon recognition. The second major aim is to dissect the molecular mechanics of mRNA recruitment to the pre-initiation complex, which is a critical event in protein synthesis and a key target of regulation. In this grant cycle, studies of mRNA recruitment will focus on the mechanisms of action of the large, heteromultimeric factor eIF3 and the ssRNA binding protein eIF4B. Preliminary work indicates central roles for both factors in this process, yet currently little is known about how they function.

Public Health Relevance

The final stage of the expression of genes is the production of the proteins that each gene encodes. When this process - called "protein synthesis" or "translation" - is performed incorrectly a variety of diseases can result, including neurodegeneration, anemia and cancer. Our studies will increase our understanding of how translation normally works, which in turn will increase our understanding of how it can break down and result in diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062128-13
Application #
8469510
Study Section
Special Emphasis Panel (ZRG1-GGG-T (02))
Program Officer
Bender, Michael T
Project Start
2000-09-11
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
13
Fiscal Year
2013
Total Cost
$404,997
Indirect Cost
$158,048
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Martin-Marcos, Pilar; Nanda, Jagpreet S; Luna, Rafael E et al. (2014) Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy. RNA 20:150-67
Saini, Adesh K; Nanda, Jagpreet S; Martin-Marcos, Pilar et al. (2014) Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex. Nucleic Acids Res 42:9623-40
Zhou, Fujun; Walker, Sarah E; Mitchell, Sarah F et al. (2014) Identification and characterization of functionally critical, conserved motifs in the internal repeats and N-terminal domain of yeast translation initiation factor 4B (yeIF4B). J Biol Chem 289:1704-22
Dong, Jinsheng; Munoz, Antonio; Kolitz, Sarah E et al. (2014) Conserved residues in yeast initiator tRNA calibrate initiation accuracy by regulating preinitiation complex stability at the start codon. Genes Dev 28:502-20
Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F et al. (2013) Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains. RNA 19:191-207
Nanda, Jagpreet S; Saini, Adesh K; Muñoz, Antonio M et al. (2013) Coordinated movements of eukaryotic translation initiation factors eIF1, eIF1A, and eIF5 trigger phosphate release from eIF2 in response to start codon recognition by the ribosomal preinitiation complex. J Biol Chem 288:5316-29
Martin-Marcos, Pilar; Nanda, Jagpreet; Luna, Rafael E et al. (2013) ?-Hairpin loop of eukaryotic initiation factor 1 (eIF1) mediates 40 S ribosome binding to regulate initiator tRNA(Met) recruitment and accuracy of AUG selection in vivo. J Biol Chem 288:27546-62
Rajagopal, Vaishnavi; Park, Eun-Hee; Hinnebusch, Alan G et al. (2012) Specific domains in yeast translation initiation factor eIF4G strongly bias RNA unwinding activity of the eIF4F complex toward duplexes with 5'-overhangs. J Biol Chem 287:20301-12
Shin, Byung-Sik; Kim, Joo-Ran; Walker, Sarah E et al. (2011) Initiation factor eIF2? promotes eIF2-GTP-Met-tRNAi(Met) ternary complex binding to the 40S ribosome. Nat Struct Mol Biol 18:1227-34
Mitchell, Sarah F; Walker, Sarah E; Algire, Mikkel A et al. (2010) The 5'-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway. Mol Cell 39:950-62

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