Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Bacterial RNA polymerase (RNAP) forms transcription-competent open complexes (RPo) via transient intermediates, but real-time structural dynamics are poorly understood.
• Understanding these intermediate steps is crucial for elucidating gene regulation mechanisms.
• The rate-limiting step in RPo formation requires overcoming significant conformational changes.

Approach:

• Utilized time-resolved cryo-electron microscopy (cryo-EM) to capture structural snapshots of intermediate states.
• Analyzed intermediates formed 120 and 500 milliseconds after mixing *Escherichia coli* σ⁷⁰-RNAP with the λP_R promoter.
• Visualized dynamic conformational changes during the initial stages of transcription initiation.

Key Points:

• Observed rapid unpairing of the upstream DNA at the promoter.
• Documented stepwise insertion of nontemplate strand bases into RNAP pockets.
• Showcased RNAP clamp closure and expulsion of the inhibitory σ⁷⁰ domain.
• Identified full template strand unpairing by 120 ms, with ongoing dynamics.

Conclusions:

• Provided the first real-time structural insights into bacterial transcription initiation intermediates.
• Demonstrated how DNA opening and nontemplate strand extrusion are critical early events.
• Highlighted the role of DNA sequence in regulating RPo formation steps, offering a generalizable model for bacterial promoters.