Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling.
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.
Montesano-Roditis
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Department of Biological Chemistry, University of California School of Medicine, Los Angeles, California 90095-1737, USA.
Glitz
D G
DG
Traut
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RR
Stewart
P L
PL
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D020285:Cryoelectron Microscopy; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D012270:Ribosomes / Q000737:chemistry* / Q000648:ultrastructure
D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D003545:Cysteine