Cold-sensitive ribosome assembly in an Escherichia coli mutant lacking a single methyl group in ribosomal protein L3

Area of Science:

• Molecular Biology
• Microbiology
• Biochemistry

Background:

• Ribosomal protein methylation is a known post-translational modification in bacteria.
• The functional significance of ribosomal protein methylation, particularly in Escherichia coli, remains largely undetermined.
• Specific methylation sites and their roles in ribosome assembly and function are areas of active investigation.

Purpose of the Study:

• To investigate the function of glutamine 150 methylation on ribosomal protein L3 in Escherichia coli.
• To characterize the phenotype of an Escherichia coli mutant (prmB2) deficient in L3 methylation.
• To assess the impact of L3 methylation on ribosomal assembly, stability, and protein synthesis activity.

Main Methods:

• Construction and analysis of an Escherichia coli prmB2 mutant lacking L3 methylation.
• Assessment of growth rates at various temperatures, particularly cold sensitivity at 22 degrees C.
• Analysis of ribosomal particle formation, stability, and protein synthesis activity in vitro and in vivo.
• In vitro methylation assays using purified components.

Main Results:

• The prmB2 mutant exhibited a cold-sensitive phenotype with significantly reduced growth rate at 22 degrees C.
• Mutant strains accumulated abnormal and unstable ribosomal particles, with reduced rates of 50-S and 30-S subunit formation.
• Despite assembly defects, purified prmB2 ribosomes demonstrated wild-type levels of protein synthesis activity in vitro.
• In vivo protein synthesis, assessed by beta-galactosidase induction, and ribosome stability under stress conditions were comparable to wild-type.

Conclusions:

• Glutamine 150 methylation of ribosomal protein L3 is essential for optimal Escherichia coli growth at low temperatures.
• While L3 methylation influences ribosome assembly and particle stability, it does not appear to affect the intrinsic catalytic activity of the assembled ribosome.
• The L3-specific methyltransferase may function as a ribosomal 'assembly factor,' facilitating efficient ribosome biogenesis under specific conditions.