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J. Biol. Chem., Vol. 276, Issue 17, 14117-14123, April 27, 2001
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From the The Escherichia coli
ribosomal protein L7/L12 is central to the translocation step of
translation, and it is known to be flexible under some conditions. The
assignment of electron density to L7/L12 was not possible in the recent
2.4 Å resolution x-ray crystallographic structure (Ban, N., Nissen,
P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000)
Science 289, 905-920). We have localized the two dimers of
L7/L12 within the structure of the 70 S ribosome using two
reconstitution approaches together with cryo-electron microscopy and
single particle reconstruction. First, the structures were determined
for ribosomal cores from which protein L7/L12 had been removed by
treatment with NH4Cl and ethanol and for reconstituted ribosomes in which purified L7/L12 had been restored to core particles. Difference mapping revealed that the reconstituted ribosomes had additional density within the L7/L12 shoulder next to protein L11.
Second, ribosomes were reconstituted using an L7/L12 variant in which a
single cysteine at position 89 in the C-terminal domain was modified
with Nanogold (Nanoprobes, Inc.), a 14 Å gold derivative. The
reconstruction from cryo-electron microscopy images and
difference mapping placed the gold at four interfacial positions. The
finding of multiple sites for the C-terminal domain of L7/L12 suggests that the conformation of this protein may change during the steps of
elongation and translocation.
Cryo-electron Microscopic Localization of Protein
L7/L12 within the Escherichia coli 70 S Ribosome by
Difference Mapping and Nanogold Labeling*
,,
Department of Biological Chemistry,
University of California School of Medicine, Los Angeles, California
90095-1737, the § Department of Biological Chemistry,
University of California School of Medicine, Davis, California 96517, and the ¶ Department of Molecular and Medical Pharmacology, Crump
Institute for Molecular Imaging, University of California School of
Medicine, Los Angeles, California 90095-1770
*
This work was supported by National Institutes of Health
Grants GM51195 (to D. G. G.) and GM 17924 (to R. T. T.) and
National Science Foundation Grant MCB-9722353 (to P. L. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed:
Dept. of Molecular and Medical Pharmacology, Crump Inst. for Molecular
Imaging, UCLA School of Medicine, A-324 CIMI, Box 951770, Los Angeles, CA 90095-1770. Tel.: 310-206-7055; Fax: 310-206-8975; E-mail: pstewart@mednet.ucla.edu.
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