Mistranslation in E. coli.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Flagellin, a key component of bacterial flagella, naturally lacks cysteine residues.
• Mistranslation, or errors in protein synthesis, can occur in vivo and impact cellular function.
• Antibiotics such as streptomycin and neomycin are known to induce errors in protein synthesis in vitro.

Purpose of the Study:

• To quantify in vivo mistranslation by measuring the incorporation of 35S-cysteine into flagellin.
• To investigate the effect of streptomycin and neomycin on flagellin mistranslation.
• To determine the probability of codon misreading during protein synthesis.

Main Methods:

• Purification of flagellin from bacterial cultures.
• Detection of 35S-cysteine incorporation into flagellin using SDS-PAGE.
• Analysis of mistranslation rates under normal and antibiotic-treated conditions.
• Investigation of arginine codon misreading and its effect on cysteine incorporation.

Main Results:

• Trace amounts of 35S-cysteine were detected in flagellin under normal conditions (approx. 6 X 10(-4) pmoles cysteine/pmole flagellin).
• Streptomycin and neomycin significantly increased 35S-cysteine incorporation into flagellin.
• Arginine starvation exacerbated cysteine incorporation in a relA- mutant, suggesting misreading of arginine codons (CGU/CGC).

Conclusions:

• In vivo mistranslation can be accurately measured by incorporating labeled amino acids into cysteine-free proteins like flagellin.
• Streptomycin and neomycin induce significant mistranslation, likely through misreading of arginine codons.
• The deduced misreading probability per codon is in the range of 10(-4).