Characterization of the chloramphenicol binding site in Escherichia coli ribosomes

Carmen Luisa Fernandez, University of Pennsylvania


Chloramphenicol (CAM) is a potent, naturally-occurring antibiotic which interferes directly with the peptidyl transferase activity of the ribosome. The interaction of CAM with E. coli ribosomes was studied by photoaffinity labeling. A variety of CAM analogues were synthesized and used to characterize the binding site of native CAM. Native CAM is shown to photoincorporate predominantly into ribosomal protein in very low yield, with little incorporation into ribosomal RNA. It is also shown that this photoincorporation is site-specific for the high affinity chloramphenicol binding site. Correct stereospecificity of photoincorporation is demonstrated by competition experiments between ($\sp3$H) -labeled CAM and excess cold CAM or excess cold LECAM (the biologically inactive isomer), and by comparison of the photoincorporation of ($\sp3$H) -CAM and ($\sp3$H) -DECAM ( ($\sp3$H) -D-erythro-chloramphenicol, also a biologically inactive isomer). Competition of ($\sp3$H) -CAM photoincorporation is effected by erythromycin, an inhibitor of high affinity CAM binding. While numerous proteins are photolabeled, only one protein group is specifically labeled. The components of this specifically labeled group were identified by a combination of chromatographic and electrophoretic techniques (RP- and IE-HPLC, 1D urea- and SDS-PAGE) as L31 and L27. It is proposed that these two proteins are components of the CAM high affinity site. Using prior cross-linking data and the results of the present study, a model for the location of L31 In the 50S subunit is proposed.

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Recommended Citation

Fernandez, Carmen Luisa, "Characterization of the chloramphenicol binding site in Escherichia coli ribosomes" (1991). Dissertations available from ProQuest. AAI9211932.