Advanced

The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain

Helgstrand, Magnus LU ; Mandava, Chandra S.; Mulder, Frans LU ; Liljas, Anders LU ; Sanyal, Suparna LU and Akke, Mikael LU (2007) In Journal of Molecular Biology 365(2). p.468-479
Abstract
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... (More)
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. (c) 2006 Elsevier Ltd. All rights reserved. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
NMR spectroscopy, GTPase, L12, ribosome, protein synthesis
in
Journal of Molecular Biology
volume
365
issue
2
pages
468 - 479
publisher
Elsevier
external identifiers
  • wos:000243243200015
  • scopus:33751532962
ISSN
1089-8638
DOI
10.1016/j.jmb.2006.10.025
language
English
LU publication?
yes
id
2a2aa3a4-6507-4f71-aaeb-73f77bb25ae6 (old id 679314)
date added to LUP
2007-12-12 12:59:39
date last changed
2017-08-27 05:11:06
@article{2a2aa3a4-6507-4f71-aaeb-73f77bb25ae6,
  abstract     = {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. (c) 2006 Elsevier Ltd. All rights reserved.},
  author       = {Helgstrand, Magnus and Mandava, Chandra S. and Mulder, Frans and Liljas, Anders and Sanyal, Suparna and Akke, Mikael},
  issn         = {1089-8638},
  keyword      = {NMR spectroscopy,GTPase,L12,ribosome,protein synthesis},
  language     = {eng},
  number       = {2},
  pages        = {468--479},
  publisher    = {Elsevier},
  series       = {Journal of Molecular Biology},
  title        = {The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain},
  url          = {http://dx.doi.org/10.1016/j.jmb.2006.10.025},
  volume       = {365},
  year         = {2007},
}