| Περίληψη: | Bacterial elongation factor P (EF-P) is a poorly understood soluble protein that has been
shown to enhance the first step of peptide bond formation through an interaction with the
ribosome and initiator tRNA. The crystal structure of EF‐P shows that EF‐P mimics the tRNA
shape. Orthologous proteins have been found in both archaeal and eukaryotic systems, known
as aIF5A and eIF5A, respectively. eIF5A, which was recently shown to increase translation
elongation rates, is post-translationally modified at a highly conserved lysine residue (K50)
through the addition of the rare amino acid hypusine. A similar pathway was recently
elucidated for EF-P, in which EF-P is post-translationally modified by the enzymes YjeA and YjeK
at lysine 34, corresponding to a homologous site of hypusylation in a/eIF5A. As a paralog of
class II LysRS, YjeA catalyzes the addition of lysine onto EF-P, but is incapable of modifying
tRNA. YjeK is a 2,3-(β)-lysine aminomutase and is responsible for converting lysine to β-lysine,
which YjeA was recently shown to recognize as a preferred substrate for EF-P modification.
However, fully modified EF-P requires a third enzyme, YfcM, which acts as a hydroxylase and
hydroxylates the C4 or C5 position of K34 of EF-P, but not the added β-lysine.
Based on a complete description of the EF-P modification and pathway, in this project
we focused on further studies to address the mechanism of action of EF-P and especially to
investigate how the different stages of EF-P’s modifications can affect E. coli cells. Using E. coli
Keio knockout collection (Δefp, ΔyjeK, ΔyjeA, ΔyfcM) and E. coli Keio parental strain (wild-type)
as reference, we checked the effect of the deletion strains on the cells under different
environmental stress conditions (varying growth temperatures and nutrition conditions,
susceptibility to antibiotics), showing that Δefp strain has growth defects and that E. coli efp
mutants show sensitivity to non-ribosomal inhibitors, such as ampicillin and rifampicin,
suggesting a possible secondary role of EF-P related to the cell envelope. Moreover, we tested
the ability of deletion strains to restore viability in the presence of the appropriate plasmid and
showed that EF-P is important for cell viability under certain conditions in E. coli. As reported
previously, YjeA and YjeK are important in bacteria virulence. In addition, EF‐P is recognized as
one of the proteins important for bacteria motility in Bacillus subtilis. However, motility and
virulence are often linked together. Here, we tested deletion strains for their ability to produce
flagella. Further, using external fluorescence staining and confocal microscopy we revealed
differences in morphology of the E. coli deletion strains, and we performed Histidine tag
protein purification with Ni-NTA agarose beads and gel filtration, in order to purify YfcM, an
uncharacterized protein, and set initial screens for crystallization. Finally, our future goal is to
clone the following polycistronic construct, “- yjeK - yjeA - yfcM - his-efp -“, overexpress and
crystallize it, so as to see the crystal structure of the whole modification pathway of EF-P and
study better the function of EF-P in translation extracts from different mutants
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