Initiation is a central point in protein synthesis. In prokaryotes, three protein initiation factors participate in assembly of the 70S initiation complex at the correct start codon. All three initiation factors are essential for cell viability, but the precise function for one, IF1, remains unclear. This proposal takes a combined structural and biochemical approach to understanding the role of IF1 in initiation. IF1 has been shown to bind within the A site of the 30S subunit, In the preliminary data, the binding site for IF1 has been localized to the end of a long helix, called the penultimate helix. A model oligonucleotide that corresponds to this helix mimics the ribosomal binding site for IF1 and forms a 1:1 complex suitable for NMR.
In specific aim 1, the three-dimensional structure of the IF1 -RNA oligonucleotide complex will be determined by NMR spectroscopy. Biophysical methods, such as titration calorimetry and NMR, will be used to probe the physical origins of specific RNA recognition by IF1.
In specific aim 2, the functional effects of IF1 binding to 30S subunits will be probed by measuring tRNA affinities and specificities for the ribosomal P-site using a gel mobility shift assay. Also, IF1 will be fluorescently labeled for binding affinity measurements for 30S and 70S ribosomes. A fluorescent label will also be incorporated into the penultimate helix by mutation to include a BIV Tat peptide binding site, which will bind fluorescent BIV Tat peptide. This will allow ribosomal conformational changes to be monitored.
In specific aim 3, the effects on IF1 binding to 30S subunits of antibiotics that disrupt A-site function will be determined. This has important implications for the mechanism of action of these drugs, and for the discovery of novel therapeutic compounds. In the final specific aim (4), the structural and functional homolog of IF1 in eukaryotic organisms will be revealed. The IF1 homolog, which is either eIF2alpha, eIF1A or eIF5A, should bind as well to the eukaryotic 40S subunit A site. Once the homolog is identified, structure determination on protein alone and on the RNA-initiation factor complex will be performed. These studies should reveal how a protein initiation factor specifically recognizes its RNA target, how ribosomal structure is changed by factor binding, and the implications of this binding for ribosome function and the ultimate expression and regulation of genetic information.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Rhoades, Marcus M
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Stanford University
Schools of Medicine
United States
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