Thermodynamic Characterization of ppGpp Binding to EF-G or 1F2 and of Initiator tRNA Binding to Free 1F2 in the Presence of GDP, GTP, or ppGpp
2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 402, no 5, 838-846 p.Article in journal (Refereed) Published
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 degrees C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 Q mu M K-d versus 9.1-13.9 mu M K-d at 10-25 degrees 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.
Place, publisher, year, edition, pages
2010. Vol. 402, no 5, 838-846 p.
IF2, EF-G, PPGPP, initiator tRNA, ITC
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-135341DOI: 10.1016/j.jmb.2010.08.016ISI: 000284182500006OAI: oai:DiVA.org:uu-135341DiVA: diva2:375489