Periera et al Paper Investigation

• summary/conclusion: YabT phosphorylates EF-Tu during sporulation to shut down protein synthesis in preparation for quiescence during sporulation
• point 1: EF-Tu and YabT are present during sporulation

  • experiment -> integrated flag epitope at end of yabT gene and had antibody for flag
  • results show
  • EF-Tu is always present throughout the cell
  • YabT appears after 2 hours of inducing sporulation

  
• point 2: YabT specifically localizes to the forespore (FS) during sporulation

  • experiment -> attached fluorescent proteins to proteins that were ONLY in mother cell (MC) or forespore
  • results -> YabT appears only in the FS not MC, EF-Tu present in both MC and FS

  
• point 3: YabT phosphorylates EF-Tu on Thr63

  • experiment -> ran western blot on samples with and without EF-Tu or YabT
  • results -> only YabT phosphorylates EF-Tu and does so on Thr63 because:
  • due to presence of double band with EF-Tu/YabT lane
  • with mutated Thr63 and YabT there was no double band

  
• point 4: phosphorylated EF-Tu can bind GTP

  • explanation -> final conclusion is that phosphorylated EF-Tu inactivates it by disabling it from dissociating from ribosome because phosphorylated EF-Tu can still bind to GTP
  • experiment -> dot blot strips of membrane containing GTP dotted with increasing concentration of EF-Tu protein species
  • WT EF-Tu
  • GTP binding deficient mutant EF-Tu
  • phosphorylated EF-Tu
  • results -> EF-Tu can bind to GTP regardless if it's phosphorylated or not

  
• point 5: phosphorylation of EF-Tu inhibits GTP hydrolysis

  • experiment -> measuring GDP levels (aka GTP hydrolysis) by thin layer chromatography (TLC) by spotting EF-Tu on membrane with solvent at bottom to wick up the membrane and separate protein by size
  • GTP, 70S ribosome (helps GTPase activity), EF-Tu by itself as control
  • WT EF-Tu
  • P~EF-Tu
  • deP~EF-Tu
  • results -> WT and dephosphorylated EF-Tu had same levels of GTP hydrolysis while phosphorylated were very low thus phosphorylation inhibits

  
• point 6: phosphorylation of EF-Tu impairs protein elongation

  • experiment -> conducted TLC by adding radioactive aa tRNA with ribosome and see if WT or phosphorylated EF-Tu have difference in levels of tripeptide or dipeptide
  • results -> WT forms tripeptide properly because EF-Tu can dissociate from ribosome but phosphorylated does not because the phosphorylation prevents EF-Tu from dissociating so it only forms a dipeptide at most