Conformational flexibility in sigma70 region 2 during transcription initiation

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Prokaryotic RNA polymerase holoenzyme, comprising core subunits and a sigma factor, regulates gene expression.
• Sigma factors, like Escherichia coli sigma(70), confer promoter specificity.
• Holoenzyme undergoes conformational changes during transcription initiation, but their impact on specific regions remains unclear.

Purpose of the Study:

• To investigate the role of conformational flexibility in sigma(70) region 2 during transcription initiation.
• To determine how restricting conformational changes in sigma(70) region 2 affects holoenzyme function.

Main Methods:

• Engineering disulfide bonds to "lock" specific subregions (2.1-2.2 and 2.2-2.3) within sigma(70) region 2.
• Characterizing mutant holoenzymes for defects in multiple-round transcription.
• Assessing the impact of disulfide bonds on open complex formation and stability.

Main Results:

• Disulfide-bonded mutant holoenzymes remained active in multiple-round transcription, showing no fundamental block.
• Both disulfide-containing holoenzymes exhibited defects in open complex formation and stability.
• These findings indicate that conformational flexibility in sigma(70) region 2 is important for these processes.

Conclusions:

• Conformational flexibility within sigma(70) region 2 is essential for efficient open complex formation.
• This flexibility facilitates the overall process of transcription initiation in prokaryotes.
• Restricting flexibility in sigma(70) region 2 negatively impacts key steps in transcription initiation.