Structural basis for transcription inhibition by E. coli SspA

Area of Science:

• Bacterial transcription regulation
• Structural biology
• Molecular mechanisms

Background:

• Stringent starvation protein A (SspA) is linked to bacterial virulence and metabolism.
• Its precise role in transcription regulation remains unclear due to limited structural data.
• Bacterial RNA polymerase (RNAP) holoenzyme structure with SspA is largely unknown.

Purpose of the Study:

• To determine the structural basis of SspA interaction with the bacterial RNAP holoenzyme.
• To elucidate the regulatory mechanism of SspA in bacterial transcription.
• To understand the σ factor selectivity of SspA.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of E. coli RNAP-promoter open complex (RPo) with SspA at 3.68 Å resolution.
• Fluorescent polarization assays to investigate SspA interactions with σ70 region 4.
• In vitro transcription assays to assess the effect of SspA on transcription initiation and elongation.

Main Results:

• The cryo-EM structure reveals SspA forms a homodimer, binding simultaneously to σ70 region 4 and the RNAP β' subunit's zinc binding domain.
• Fluorescent polarization data confirm specific interactions between SspA and σ70 region 4, conferring σ factor selectivity.
• In vitro transcription assays demonstrate that SspA inhibits transcription, likely by suppressing promoter escape.

Conclusions:

• The study provides the first structural insights into SspA bound to the bacterial RNAP holoenzyme.
• SspA's homodimeric binding and interaction with σ70 region 4 explain its σ factor selectivity.
• SspA inhibits bacterial transcription by suppressing promoter escape, offering a new understanding of its physiological role.