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.
Department of Biological Chemistry, University of California School of Medicine, Los Angeles, California 90095-1737, USA.
L
L
Montesano-Roditis
D G
DG
Glitz
R R
RR
Traut
P L
PL
Stewart
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