Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• mRNA translation speed is influenced by amino acid incorporation, with proline and adjacent prolines causing significant ribosome stalling.
• Previous research focused on amino acid context, but the role of mRNA and codon usage in proline-induced stalling was less understood.

Purpose of the Study:

• To investigate the impact of mRNA codon choice and tRNA availability on ribosome pausing at proline-rich sequences.
• To explain genome-scale observations of proline codon usage in Escherichia coli.

Main Methods:

• Bioinformatic analysis of the Escherichia coli genome to identify codon usage patterns for proline.
• Development and utilization of a luminescence reporter system in living cells to measure ribosome pausing.
• Dissection of the roles of specific codon choices and cognate tRNA levels in regulating translation efficiency and pausing.

Main Results:

• Significant differences in codon usage were observed between single and consecutive proline codons in the E. coli genome.
• The CCC/U-C codon sequon was found to strongly select against due to ribosomal frameshifting.
• The CCG codon is overrepresented, translated fastest, and associated with near-saturating prolyl-tRNA levels, indicating selection for translation efficiency.
• The CCA codon, with limiting prolyl-tRNA, is utilized to fine-tune ribosome pausing strength.

Conclusions:

• Codon selection, particularly for proline pairs, is a critical mechanism for tuning protein expression levels.
• Evolutionary pressures favor efficient translation through specific codon choices, while others are selected against due to detrimental effects like frameshifting.
• Ribosome pausing and translation efficiency are dynamically regulated by the interplay of codon usage and tRNA availability.