Elongation Factor P (EF-P) is a universally conserved post-translationally modified protein that relieves ribosomal pausing at polyproline motifs by binding to the ribosome and entropically stimulating peptide bond formation. In all examples characterized to date EF-P and its homologs require post-translational modification to be functional. The function of EF-P modifications and the mechanism by which modifications would improve translation efficiency is unclear. Mutations in bacterial genes encoding EF-P (efp) or the corresponding modification pathways are highly pleiotropic, leading to a variety of detrimental phenotypes including slowed growth, loss of motility, attenuated virulence and hypersensitivity to antibiotics. Preliminary characterization of post-translational modification of Bacillus subtilis EF-P, which requires 5-aminopentanol addition for activity, challenges the notion that EF-P functions solely to maintain basic cellular function. In B. subtilis the ratio of modified to unmodified EF-P varies with growth phase, and neither deletion of EF-P nor removal of the modification impair vegetative growth, but instead specifically impair motility development. We hypothesize, based on the chemical diversity of permissive post-translational modification groups, that EF-P modification is regulatory. B. subtilis is an ideal model organism to explore this hypothesis as defects in either EF-P or EF-P modification impair swarming motility, a powerful phenotype for unbiased genetic selection. The objectives of this proposal are to uncover the structural and functional diversity of EF-P by investigating the different mechanisms by which this conserved, ubiquitous, translation factor functions during protein synthesis. Specifically, we will determine how EF-P acts as a cellular differentiation-specific translation factor and investigate the mechanism of translational control by EF-P.
Infections with pathogenic isolates of Salmonella, E. coli, Pseudomonas sp. and Bacillus sp. can all lead to infectious diseases with potentially fatal sequelae. EF-P proteins contribute to the pathogenicity of the causative agents of these and other diseases by controlling the translation of proteins critical for modulating antibiotic resistance, motility and other traits that play key roles in establishing virulence. The proposed studies will define how EF-P functions in bacterial translational control, and may provide indicators as to whether corresponding pathways, which are unique to bacteria, can be used as targets for anti-infective agents. eIF5A, the eukaryotic homolog of EF-P, requires modification with the amino acid hypusine to function in translation and has been implicated in promoting translation of mRNAs encoding proteins involved in cell cycle progression and inflammation, linking this factor to both cancer and diabetes. The proposed investigations on how EF-P functions in protein synthesis and translational control will provide new insights into the roles EF-P, and indirectly eIF5a, play in regulating gene expression.
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