The distance between two functionally significant regions of the 50 S Escherichia coli ribosome: the erythromycin binding site and proteins L7 L12

Richard Langlois, C. C. Lee, Charles R. Cantor, Robert Vince, Sidney Pestka

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The distance between the erythromycin binding site on the 50 S Escherichia coli ribosome and protein L7 has been measured by singlet-singlet energy transfer. A non-covalently bound erythromycin derivative, fluoroscein isothiocyanate erythromycylamine, was used as the acceptor. This derivative can be completely displaced from ribosomes by erythromycin, suggesting that they have the same binding site. 1,5-Iodoacetylethylenediamine naptholsulfonate-labeled protein L7 served as the fluorescent donor. It was reconstituted with salt/ethanol-washed 50 S cores. This readdition was accompanied by total recovery of elongation factor G-dependent GTPase activity. This suggests that the protein modification does not significantly perturb 50 S function or structure. Energy transfer measurements by both static and lifetime techniques were in good agreement. After consideration of various errors that enter the measurements and calculations, the L7-erythromycin distance is estimated to be 70 ± 10 Å. This long distance is interesting, since both sites may be involved in translocation. The fluorescent derivative of erythromycin was also used to study binding kinetics to the 50 S and 70 S ribosomes. Binding is a simple second-order step and proceeds about 11 times faster on the 70 S particle. Exchange of the fluorescent derivative with excess erythromycin is limited by the dissociation rate, and this is four times faster on the 70 S particle. These results suggest that the erythromycin site is more accessible on the 70 S particle, and may be an indication of conformational changes in the 50 S ribosome upon combination with the 30 S ribosome.

Original languageEnglish (US)
Pages (from-to)297-313
Number of pages17
JournalJournal of Molecular Biology
Issue number2
StatePublished - Sep 15 1976
Externally publishedYes

Bibliographical note

Funding Information:
\Ve thank Alice Beekman and Arthur .Johnson for assistance in the ribosome preparations and Robert Fairclougll for the preparation of I-13H]AEDANS. This work was supported in part by United States Public Health Services research grants GM19843 and GM14825 to one of us (C. K. (I.). One autllor (R. V.) gratefully acknowledges support by a Research Career Development Award (CA25258) from the Nationa. Cancer Institute.


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