Expression of Escherichia coli pabA

Area of Science:

• Microbiology
• Molecular Biology
• Gene Regulation

Background:

• Escherichia coli pabA gene encodes a subunit of p-aminobenzoate synthase, essential for folate biosynthesis.
• p-aminobenzoate synthase activity is crucial for bacterial survival and involves multiple enzymatic steps.
• Previous studies identified two promoters (P1 and P2) for pabA transcription, but only P1 showed expression.

Purpose of the Study:

• To investigate the regulatory mechanisms controlling pabA gene expression from its two identified promoters (P1 and P2).
• To understand why the P2 promoter does not contribute to pabA expression despite its presence.
• To identify factors influencing pabA transcription and translation initiation.

Main Methods:

• Construction and analysis of pabA-lacZ translational fusions in Escherichia coli.
• Assessment of gene expression under various conditions including nutrient limitation, growth rate changes, and genetic manipulations.
• RNA secondary structure analysis and mutational studies of the intergenic region between fic and pabA.

Main Results:

• pabA expression from a chromosomal pabA-lacZ fusion was not affected by p-aminobenzoate levels, growth rate, catabolite repression, or gene dosage.
• The P2 promoter's lack of contribution to pabA expression was attributed to a stable RNA secondary structure in the intergenic region.
• Disruption of this secondary structure through mutation or ribosome translation into the region restored pabA expression from P2.

Conclusions:

• A stable RNA secondary structure in the fic-pabA intergenic region sequesters the pabA ribosome binding site, inhibiting translation initiation from P2.
• This structural element acts as a regulatory mechanism to control pabA expression.
• Modifying this secondary structure provides a means to activate P2-driven pabA expression, offering insights into bacterial gene regulation.