Abstract
Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(β)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the β-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational β-lysylation but not hydroxylation.
Highlights
Post-translational modification activates bacterial elongation factor P (EF-P) in several Gram-negative bacteria
Polysome Profiles Indicate a Role for EF-P in Translation Elongation—Polysomes were isolated and fractionated from E. coli wild type and ⌬efp strains to investigate at which stages in translation this factor might function
There has been no other biochemical evidence to directly support a role for EF-P in initiation, whereas it was recently shown that EF-P facilitates synthesis of stretches of proline residues during translation elongation [5, 6]
Summary
Post-translational modification activates bacterial elongation factor P (EF-P) in several Gram-negative bacteria. Post-translational modification of bacterial elongation factor P (EF-P) with (R)--lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. 4416 JOURNAL OF BIOLOGICAL CHEMISTRY between the P and E sites while making several interactions with the 3Ј acceptor stem of a P-site-bound initiator tRNA [4] Based on these structural data and other functional studies, it was proposed that EF-P promotes formation of the first peptide bond during protein synthesis. In S. enterica, disruptions of the genes encoding EF-P or the -lysyl modification pathway result in a wide range of phenotypic changes including increased sensitivity to several antibiotics, loss of motility, attenuated virulence, and sensitivity to detergents and low osmolarity conditions [8] Many of these phenotypes are consistent with increased membrane permeability, suggesting a role for EF-P in regulating expression of outer membrane proteins, such as KdgM [3]. The possible role of YfcM-catalyzed hydroxylation in modulating EF-P activity is discussed
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