Single-stranded DNA drives σ subunit loading onto mycobacterial RNA polymerase to unlock initiation-competent...

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Transcription initiation is a fundamental biological process regulated by RNA polymerase (RNAP) and its sigma (σ) subunit.
• Formation of the open promoter complex (RPo) involves DNA unwinding, a critical step whose precise mechanism is not fully understood.
• Bacterial RNAP utilizes the σ subunit to recognize promoter -10 elements and initiate DNA unwinding.

Purpose of the Study:

• To investigate the conformational changes in Mycobacterium tuberculosis RNAP upon binding to a -10 single-stranded DNA (-10 ssDNA) fragment.
• To elucidate the role of the σ subunit in mediating RNAP conformational dynamics during transcription initiation.
• To reveal the mechanism of ssDNA-guided RNAP maturation.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes.
• Cryo-electron microscopy (cryo-EM) to determine high-resolution structures.
• Biochemical assays to study protein-DNA interactions.

Main Results:

• -10 ssDNA binding induced σ subunit loading onto RNAP's DNA/RNA channels.
• This binding event triggered RNAP clamp closure and unswiveling, essential for RPo formation.
• The σ subunit was identified as a key regulator of RNAP conformational dynamics.

Conclusions:

• Single-stranded DNA acts as a guide to promote RNAP maturation and conformational changes necessary for transcription initiation.
• The σ subunit plays a critical role in regulating RNAP conformational dynamics in response to DNA binding.
• This study provides mechanistic insights into transcription initiation in Mycobacterium tuberculosis.