Chloramphenicol Interferes with 50S Ribosomal Subunit Maturation via Direct and Indirect Mechanisms

Area of Science:

• Molecular Biology
• Microbiology
• Structural Biology

Background:

• Chloramphenicol (CAM) is a broad-spectrum antibiotic inhibiting bacterial protein synthesis by targeting ribosomes.
• Previous studies suggest CAM disrupts ribosome assembly by altering ribosomal protein balance.

Purpose of the Study:

• To investigate the detailed effects of CAM on the maturation process of the 50S ribosomal subunit in Escherichia coli.
• To elucidate the mechanisms by which CAM interferes with ribosome assembly.

Main Methods:

• Label-free quantitative mass spectrometry (LFQ-MS) to analyze protein expression changes.
• Cryo-electron microscopy (cryo-EM) with heterogeneous reconstruction to identify and characterize pre-50S intermediates.
• Enzyme-linked immunosorbent assay (ELISA) to detect direct binding of CAM to ribosomal precursors.

Main Results:

• CAM treatment upregulates the expression of ribosomal assembly factors.
• Cryo-EM identified 26 distinct pre-50S intermediates, revealing nine major structural states.
• CAM significantly hinders the formation of the central protuberance (CP), H89, and H58 during 50S subunit assembly.
• ELISA confirmed direct binding of CAM to ribosomal precursors.

Conclusions:

• CAM interferes with 50S ribosomal subunit maturation through a combination of direct binding and indirect effects on assembly factors.
• The study provides novel insights into CAM's mechanism of action and the complex landscape of ribosome assembly.