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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
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
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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
Abstract [en]

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.
Keyword [en]
IF2, EF-G, PPGPP, initiator tRNA, ITC
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-135341DOI: 10.1016/j.jmb.2010.08.016ISI: 000284182500006OAI: oai:DiVA.org:uu-135341DiVA: diva2:375489
Available from: 2010-12-08 Created: 2010-12-06 Last updated: 2010-12-08Bibliographically approved

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