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.
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.
Mitkevich
Vladimir A
VA
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St. 32, Moscow 119991, Russia.
Ermakov
Andrey
A
Kulikova
Alexandra A
AA
Tankov
Stoyan
S
Shyp
Viktoriya
V
Soosaar
Aksel
A
Tenson
Tanel
T
Makarov
Alexander A
AA
Ehrenberg
Mans
M
Hauryliuk
Vasili
V
D002151:Calorimetry; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D039881:Prokaryotic Initiation Factor-2 / Q000378:metabolism*; D011485:Protein Binding; D012358:RNA, Transfer, Met / Q000378:metabolism*; D013816:Thermodynamics
D020653:Peptide Elongation Factor G; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet); D006153:Guanosine Diphosphate; D006159:Guanosine Tetraphosphate; D006160:Guanosine Triphosphate