Thermodynamic characterization of ppGpp binding to EF-G or IF2 and of initiator tRNA binding to free IF2 in the presence of GDP, GTP, or ppGpp

J Mol Biol. 2010 Oct 8;402(5):838-46. doi: 10.1016/j.jmb.2010.08.016. Epub 2010 Aug 14.


In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K(d)versus 9.1-13.9 μM K(d) at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA(fMet) demonstrating that IF2 binds to initiator tRNA with submicromolar K(d) and that affinity is altered by the G nucleotides only slightly. This--in conjunction with earlier reports on IF2 interactions with fMet-tRNA(fMet) in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA(fMet) binding and GTP was suggested to strongly promote fMet-tRNA(fMet) binding--sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA(fMet) selection.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calorimetry
  • Guanosine Diphosphate / metabolism*
  • Guanosine Tetraphosphate / metabolism*
  • Guanosine Triphosphate / metabolism*
  • Kinetics
  • Peptide Elongation Factor G / metabolism*
  • Prokaryotic Initiation Factor-2 / metabolism*
  • Protein Binding
  • RNA, Transfer, Met / metabolism*
  • Thermodynamics


  • Peptide Elongation Factor G
  • Prokaryotic Initiation Factor-2
  • RNA, Transfer, Met
  • fMet-tRNA(fMet)
  • Guanosine Diphosphate
  • Guanosine Tetraphosphate
  • Guanosine Triphosphate