A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The sigma70 (σ70) initiation factor is crucial for bacterial transcription initiation in Escherichia coli.
• Regulation of phage gene expression often involves precise control of transcription elongation.
• Promoter-proximal pausing is a known mechanism for regulating gene expression.

Purpose of the Study:

• To investigate the role of the Escherichia coli σ70 initiation factor in transcription pausing.
• To identify the specific sequences and conditions that induce transcription pausing during phage gene expression.
• To elucidate the mechanism by which σ70 binding and DNA scrunching contribute to pausing.

Main Methods:

• In vitro transcription assays using purified components.
• Site-directed mutagenesis to alter promoter sequences and σ70 binding sites.
• Analysis of paused transcription complexes using biochemical methods.

Main Results:

• Escherichia coli σ70 factor induces a promoter-proximal transcription pause critical for lambdoid phage late gene regulation.
• The pause is triggered by σ70 binding to a -10 sequence repeat and DNA scrunching.
• A specific DNA sequence, the 'elemental pause sequence,' is identified as the pause site within the scrunched complex.
• Both σ70 binding and the elemental pause sequence are required to establish a significant pause; neither alone is sufficient.

Conclusions:

• The σ70 initiation factor plays a dual role in transcription initiation and elongation control through pausing.
• The interaction between σ70, the -10 promoter element, and the elemental pause sequence dictates the precise location and stability of the transcription pause.
• This mechanism provides a detailed insight into the regulation of phage gene expression at the transcriptional level.