PMID:11278411
| Citation |
Montesano-Roditis, L, Glitz, DG, Traut, RR and Stewart, PL (2001) Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling. J. Biol. Chem. 276:14117-23 |
|---|---|
| Abstract |
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 A 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 NH(4)Cl 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 A 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. |
| Links |
PubMed Online version:10.1074/jbc.M008430200 |
| Keywords |
Binding Sites; Chromatography, High Pressure Liquid; Cryoelectron Microscopy; Cysteine/chemistry; Escherichia coli/chemistry; Image Processing, Computer-Assisted; Models, Molecular; Protein Biosynthesis; Protein Conformation; Protein Structure, Tertiary; Ribosomal Proteins/chemistry; Ribosomal Proteins/ultrastructure; Ribosomes/chemistry; Ribosomes/ultrastructure |
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