Systematic search for the Cra-binding promoters using genomic SELEX system

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• The global transcription factor Cra (Catabolite Repressor Activator) regulates glycolysis and gluconeogenesis genes in E. coli.
• Understanding the full network of Cra-mediated transcriptional regulation is crucial for metabolic studies.

Purpose of the Study:

• To identify novel Cra-binding sequences and understand the transcriptional regulatory network controlled by Cra in the E. coli genome.
• To investigate the binding affinity differences between Cra-mediated repression and activation.

Main Methods:

• Genomic SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was employed to isolate Cra-binding sequences.
• Quantitative gel shift assays were used to determine binding affinities.
• In vivo promoter strength assays using a two-fluorescent protein (TFP) vector and in vitro transcription assays confirmed gene regulation.

Main Results:

• Ninety-seven Cra-binding sequences were identified, mapping to ten genomic regions in E. coli.
• Six known and four novel gene promoters were identified as Cra-binding sites.
• All identified known promoters were repressed by Cra, with higher binding affinity compared to activation-type promoters.
• Two newly identified Cra-binding sequences are associated with the gapA and eno genes involved in sugar metabolism.

Conclusions:

• Cra exhibits a stronger binding affinity for repression-type promoters, explaining the underrepresentation of activation-type promoters in SELEX.
• The study expands the known regulatory network of Cra, identifying new targets involved in sugar metabolism.
• Novel methods confirmed the regulatory roles of Cra on newly identified genes, providing a deeper understanding of metabolic control in E. coli.