Structural insights into human Pol III transcription initiation in action

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• RNA polymerase III (Pol III) transcribes essential small RNAs using classical promoters.
• The transition mechanism from Pol III preinitiation complex (PIC) to elongation complex (EC) remains poorly understood.
• Previous studies determined structures of Pol III PIC and EC but lacked insight into the initiation-elongation switch.

Purpose of the Study:

• To elucidate the molecular mechanism of the transition from Pol III initiation to elongation.
• To capture and characterize intermediate states of human Pol III transcribing complexes.
• To understand the dynamics of transcription bubble, general transcription factor (GTF) dissociation, and promoter escape.

Main Methods:

• Reconstitution of seven human Pol III transcribing complexes (TCs) halted on U6 promoters.
• Cryo-electron microscopy (cryo-EM) to determine structures of initially transcribing complexes (ITCs) and ECs.
• KMnO4 footprinting to analyze structural rearrangements and DNA-RNA hybrid dynamics.

Main Results:

• Cryo-EM structures revealed extensive modular rearrangements during the ITC-EC transition.
• The transcription bubble expands until TC5, then collapses abruptly from TC5 to TC6 with GTF dissociation.
• SNAPc and TFIIIB remain bound in TC5, with BRF2 blocking template DNA; hybrid translocation triggers GTF release and bubble collapse.

Conclusions:

• The study reveals the earliest documented initiation-elongation transition for any RNA polymerase.
• Molecular insights into Pol III dynamics, including BRF2-finger retraction and promoter escape, are provided.
• Findings shed light on Pol III reinitiation mechanisms on type 3 promoters for highly demanded small RNAs.