title: 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.
abstract: 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.
authors: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov St. 32, Moscow 119991, Russia.
authors: Vladimir A
authors: VA
authors: Mitkevich
authors: Andrey
authors: A
authors: Ermakov
authors: Alexandra A
authors: AA
authors: Kulikova
authors: Stoyan
authors: S
authors: Tankov
authors: Viktoriya
authors: V
authors: Shyp
authors: Aksel
authors: A
authors: Soosaar
authors: Tanel
authors: T
authors: Tenson
authors: Alexander A
authors: AA
authors: Makarov
authors: Mans
authors: M
authors: Ehrenberg
authors: Vasili
authors: V
authors: Hauryliuk
mesh_terms: 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
chemical_list: 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