High-resolution mapping of sigma factor DNA-binding sequences using artificial promoters, RNA aptamers, and deep...

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The sigma (σ) subunit of bacterial RNA polymerase is essential for promoter recognition and transcription initiation.
• Understanding σ-factor DNA-binding sequences is critical for deciphering bacterial gene regulation and controlling gene expression.

Purpose of the Study:

• To develop and apply a data-driven, high-throughput method for discovering σ-factor DNA-binding motifs.
• To expand the known repertoire of σ54 DNA-binding sequences in Pseudomonas putida.

Main Methods:

• Generation of a large DNA library (1.54 million templates) with artificial promoters and 5' untranslated regions.
• In vitro transcription, RNA aptamer selection, and deep DNA/RNA sequencing to assess promoter activity and quantify mRNA levels.
• Application to map σ54 DNA-binding sequences in Pseudomonas putida.

Main Results:

• Identification of 64,966 distinct σ54 binding motifs, significantly expanding the known sequence repertoire.
• Direct assessment of promoter function and quantification of promoter strength based on mRNA production.
• Demonstrated a data-driven approach that surpasses traditional methods by evaluating promoter function directly and avoiding binding affinity bias.

Conclusions:

• The developed high-throughput method is effective for discovering novel σ-factor binding motifs and characterizing promoter activity.
• The comprehensive dataset of σ54 binding sequences provides valuable insights into bacterial gene regulation.
• This approach opens new avenues for research in molecular biology and biotechnology applications.