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Class-1 release factor eRF1 promotes GTP binding by class-2 release factor eRF3
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Ehrenberg)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Ehrenberg)
2006 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 88, no 7, 747-757 p.Article in journal (Refereed) Published
Abstract [en]

In eukaryotes, termination of mRNA translation is triggered by the essential polypeptide chain release factors eRF1, recognizing all three stop codons, and eRF3, a member of the GTPase superfamily with a role that has remained opaque. We have studied the kinetic and thermodynamic parameters of the interactions between eRF3 and GTP, GDP and the non-hydrolysable GTP analogue GDPNP in the presence (K-D(GDP) = 1.3 +/- 0.2 mu M, K-D(GTP) approximate to 200 mu M and K-D(GDPNP) > 160 mu M) as well as absence (K-D(GDP) = 1.9 +/- 0.3 mu M, K-D(GTP) 0.7 +/- 0.2 mu M and K-D(GDPNP) approximate to 200 mu M) of eRF1. From the present data we propose that (i) free eRF3 has a strong preference to bind GDP compared to GTP (ii) eRF3 in complex with eRF1 has much stronger affinity to GTP than free eRF3 (iii) eRF3 in complex with PABP has weak affinity to GTP (iv) eRF3 in complex with eRF1 does not have strong affinity to GDPNP, implying that GDPNP is a poor analogue of GTP for eRF3 binding.

Place, publisher, year, edition, pages
2006. Vol. 88, no 7, 747-757 p.
Keyword [en]
GTPase, eRF1, eRF3, PABP, GDPNP, GEF, eukaryotes, translation termination, polypeptide release factors
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-97261DOI: 10.1016/j.biochi.2006.06.001ISI: 000240167100001PubMedID: 16797113OAI: oai:DiVA.org:uu-97261DiVA: diva2:172120
Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2011-06-20Bibliographically approved
In thesis
1. A Few Strokes to the Family Portrait of Translational GTPases
Open this publication in new window or tab >>A Few Strokes to the Family Portrait of Translational GTPases
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work.

In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction.

The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event.

Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 86 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 435
Molecular biology, translation, GTPase, EF-G, eRF3, Molekylärbiologi
urn:nbn:se:uu:diva-8844 (URN)978-91-554-7210-8 (ISBN)
Public defence
2008-05-28, B21, ICM BMC, Husargatan 3, Uppsala, 13:00
Available from: 2008-05-07 Created: 2008-05-07Bibliographically approved

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Hauryliuk, VasiliEhrenberg, Måns
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