Related Experiment Video
Updated: Jul 24, 2026

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
Insights into the mechanism of azithromycin interaction with an Escherichia coli functional ribosomal complex
G P Dinos1, M Michelinaki, D L Kalpaxis
1Laboratory of Biochemistry, School of Medicine, University of Patras, GR-26500 Patras, Greece.
Abstract:
Azithromycin, a derivative of erythromycin with improved activity against Gram-negative bacteria, exhibits a marginal inhibition effect in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. This renders the study of azithromycin interaction with Ac[(3)H]Phe-tRNA. poly(U). 70S ribosome complex (complex C) impossible, if we analyze its effect on peptide bond formation. To overcome this problem, we have used an alternative approach to investigate kinetically the azithromycin interaction with complex C and to compare the azithromycin binding properties with those of erythromycin. This approach was based on the ability of azithromycin to compete with tylosin, a macrolide antibiotic strongly inhibiting the puromycin reaction. Detailed kinetic analysis revealed that the encounter complex CA between complex C and azithromycin (A) undergoes a slow isomerization to a tighter complex C*A, which remains active toward puromycin. The determination of inhibition and isomerization rate constants enabled us to classify azithromycin as a slow-binding ligand of ribosomes. Compared with erythromycin, azithromycin is a better inducer and stabilizer of the C*A complex. This finding may explain the superiority of azithromycin as inhibitor of translation in E. coli cells and many other Gram-negative bacteria.
More Related Videos
14:58Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
Published on: November 12, 2012
10:41The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli
Published on: January 13, 2013
Related Concept Videos
Types of RNA
Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use.
RNA...
Improving Translational Accuracy
Coordination of Gene Expression Processes in Bacteria
Stringent Response in E. coli
Mechanism of Antibiotic Resistance in MRSA
Inhibitors of Bacterial Protein Synthesis