uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Show others and affiliations
2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 2, 468-479 p.Article in journal (Refereed) Published
Abstract [en]

Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.

Place, publisher, year, edition, pages
2007. Vol. 365, no 2, 468-479 p.
Keyword [en]
L12, ribosome, GTPase, NMR spectroscopy, protein synthesis
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-146545DOI: 10.1016/j.jmb.2006.10.025ISI: 000243243200015PubMedID: 17070545OAI: oai:DiVA.org:uu-146545DiVA: diva2:398408
Available from: 2011-02-17 Created: 2011-02-17 Last updated: 2011-11-03Bibliographically approved
In thesis
1. Ribosomal Stalk Protein L12: Structure, Function and Application
Open this publication in new window or tab >>Ribosomal Stalk Protein L12: Structure, Function and Application
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribosomal stalk proteins are known to play important role in protein synthesis. The ‘stalk’, an extended structure on the large subunit of the ribosome is composed mainly of two to three dimers of L12 and one L10 protein, which forms the base of the stalk. In E. coli, four copies of L12 molecules exist as dimer of dimers forming the pentameric L8 complex together with L10. This thesis is a collection of four interlinked studies on the structure, function and application of the ribosomal stalk protein L12. In the first study, we have mapped the interaction sites of the four major translation GTPase factors (IF2, EF-Tu, EF-G & RF3) on L12 molecule using heteronuclear NMR spectroscopy. Surprisingly, all these factors produced an overlapping interaction map spanning two α-helices on the C terminal domain of L12, thereby suggesting a general nature of the interaction between L12 and the GTPase factors. L12 is known to stimulate GTPase activity of the elongation factors EF-Tu and EF-G. Here, we have clarified the role of L12 in IF2 mediated initiation of protein synthesis. Our data suggest that rapid subunit association requires a specific interaction between the L12 protein on the 50S and IF2·GTP on the 30S preinitiation complex. We have also shown that L12 is not a GAP for IF2 and GTP hydrolysis triggers IF2 release from the 70S initiation complex. The next question we have addressed is why multiple copies of L12 dimer are needed on the ribosome. For this purpose, we created a pure E. coli strain JE105, where the terminal part of rplJ gene coding for the binding site of one L12 dimer on protein L10 was deleted in the chromosomal locus. Using ribosomes with single L12 dimer we have observed that the rate of the initiation and elongation involving IF2 and EF-G gets most compromised, which in turn decreases the growth rate of the bacteria.  This study also indicates that L12 can interact with different GTPase factors in a specialized manner. Lastly, we have developed an application making advantage of the multiple L12 dimers on the ribosome. By inserting a (His)6-tag at the C-terminus of the L12 protein we have created a novel E. coli strain (JE28), where all ribosomes are tetra-(His)6-tagged. Further, we have developed a single step method for purification of the active (His)6-tagged ribosomes from JE28.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 848
Ribosome, protein synthesis, L12 dimer, initiation, elongation, G factors, and His-tag
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
urn:nbn:se:uu:diva-157198 (URN)978-91-554-8144-5 (ISBN)
Public defence
2011-10-06, B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2011-09-15 Created: 2011-08-19 Last updated: 2011-11-03Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed
By organisation
Department of Cell and Molecular Biology
In the same journal
Journal of Molecular Biology
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

Total: 142 hits
ReferencesLink to record
Permanent link

Direct link