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
Mitkevich, Vladimir A. ; Ermakov, Andrey ; Kulikova, Alexandra A. ; Tankov, Stoyan ; Shyp, Viktoriya ; Soosaar, Aksel ; Tenson, Tanel ; Makarov, Alexander A. ; Ehrenberg, Mans and Hauryliuk, Vasili LU
(2010)
In Journal of Molecular Biology
402(5).
p.838-846
- 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 Kd versus 9.1-13.9 μM Kd 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-tRNAfMet demonstrating that IF2... (More)
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 Kd versus 9.1-13.9 μM Kd 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-tRNAfMet demonstrating that IF2 binds to initiator tRNA with submicromolar Kd and that affinity is altered by the G nucleotides only slightly. This-in conjunction with earlier reports on IF2 interactions with fMet-tRNAfMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNAfMet binding and GTP was suggested to strongly promote fMet-tRNAfMet binding-sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNAfMet selection.
(Less)
- author
- Mitkevich, Vladimir A.
; Ermakov, Andrey
; Kulikova, Alexandra A.
; Tankov, Stoyan
; Shyp, Viktoriya
; Soosaar, Aksel
; Tenson, Tanel
; Makarov, Alexander A.
; Ehrenberg, Mans
and Hauryliuk, Vasili
LU
- publishing date
- 2010-10-08
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- EF-G, IF2, Initiator tRNA, ITC, PpGpp
- in
- Journal of Molecular Biology
- volume
- 402
- issue
- 5
- pages
- 838 - 846
- publisher
- Elsevier
- external identifiers
-
- pmid:20713063
- scopus:77957233585
- ISSN
- 0022-2836
- DOI
- 10.1016/j.jmb.2010.08.016
- language
- English
- LU publication?
- no
- additional info
- Funding Information: We are grateful to Michael Cashel for sharing the overexpression construct for the Rel/Spo enzyme from S. equisimilisas and the detailed protocols for ppGpp purification. We are also grateful to Pohl Milon for helpful discussions and to TaylanBeyaz for assistance with ppGpp purification. This work was supported by the Presidium of the Russian Academy of Sciences (Program Molecular and Cell Biology to A.A.M.); Estonian Science Foundation grants ( 6768 to T.T. and 7616 to V.H.); National Institutes of Health grant RO1 GM070768 ; the Swedish Research Council (M.E.); the Russian Foundation for Basic Research ( 10-04-01746-а to V.A.M.); the Dmitry Zimin Dynasty Foundation (A.A.K.); and the European Regional Development Fund through the Center of Excellence in Chemical Biology (V.H. and T.T.). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.
- id
- 6534a1d0-2902-457f-a495-085292a6d8ff
- date added to LUP
- 2021-09-24 20:49:06
- date last changed
- 2024-01-05 15:59:43
@article{6534a1d0-2902-457f-a495-085292a6d8ff,
abstract = {{<p>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<sub>d</sub> versus 9.1-13.9 μM K<sub>d</sub> 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<sup>fMet</sup> demonstrating that IF2 binds to initiator tRNA with submicromolar K<sub>d</sub> and that affinity is altered by the G nucleotides only slightly. This-in conjunction with earlier reports on IF2 interactions with fMet-tRNA<sup>fMet</sup> in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA<sup>fMet</sup> binding and GTP was suggested to strongly promote fMet-tRNA<sup>fMet</sup> binding-sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA<sup>fMet</sup> selection.</p>}},
author = {{Mitkevich, Vladimir A. and Ermakov, Andrey and Kulikova, Alexandra A. and Tankov, Stoyan and Shyp, Viktoriya and Soosaar, Aksel and Tenson, Tanel and Makarov, Alexander A. and Ehrenberg, Mans and Hauryliuk, Vasili}},
issn = {{0022-2836}},
keywords = {{EF-G; IF2; Initiator tRNA; ITC; PpGpp}},
language = {{eng}},
month = {{10}},
number = {{5}},
pages = {{838--846}},
publisher = {{Elsevier}},
series = {{Journal of Molecular Biology}},
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}},
url = {{http://dx.doi.org/10.1016/j.jmb.2010.08.016}},
doi = {{10.1016/j.jmb.2010.08.016}},
volume = {{402}},
year = {{2010}},
}