Structural basis of TFIIIC-dependent RNA Polymerase III transcription initiation

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• RNA Polymerase III (Pol III) transcribes essential non-coding RNAs, including 5S ribosomal RNA (rRNA) and tRNAs.
• Transcription initiation by Pol III requires specific transcription factors (TFIIIA, TFIIIC, TFIIIB) to bind the 5S rRNA promoter.
• Understanding the precise mechanism of transcription factor recruitment and DNA interaction is crucial for regulating Pol III activity.

Approach:

• Utilized cryo-electron microscopy (cryo-EM) to determine the high-resolution structure of the S. cerevisiae TFIIIA-TFIIIC-promoter complex.
• Investigated the role of Brf1-TBP binding in stabilizing the DNA-bound transcription initiation complex.
• Employed single-molecule Förster Resonance Energy Transfer (smFRET) to study DNA dynamics, including bending and dissociation, during complex assembly.

Key Points:

• Visualized the S. cerevisiae TFIIIA and TFIIIC complex bound to the 5S rRNA promoter.
• Observed full-length 5S rRNA gene wrapping around the complex upon Brf1-TBP stabilization.
• smFRET data revealed slow-timescale DNA bending and partial dissociation, consistent with cryo-EM structural models.

Conclusions:

• Provides unprecedented structural insights into the assembly of the Pol III transcription initiation complex.
• Elucidates the dynamic DNA interactions essential for 5S rRNA gene transcription.
• Offers a mechanistic understanding of a critical step in Pol III transcription regulation.