Structural basis of TFIIIC-dependent RNA polymerase III transcription initiation

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• RNA polymerase III (Pol III) transcribes essential non-coding RNAs like 5S rRNA and tRNAs.
• Assembly of the Pol III transcription initiation complex requires specific transcription factors: TFIIIA, TFIIIC, and TFIIIB.
• Understanding the structural basis of this assembly is crucial for deciphering gene regulation.

Purpose of the Study:

• To determine the high-resolution structure of the Saccharomyces cerevisiae TFIIIA-TFIIIC-promoter complex.
• To visualize the DNA binding of TFIIIB subunits (Brf1 and TBP) within the transcription initiation complex.
• To investigate the dynamic behavior of DNA during transcription initiation.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to resolve the complex structure.
• Single-molecule Förster Resonance Energy Transfer (smFRET) to study DNA dynamics.
• Biochemical assays to analyze protein-DNA interactions.

Main Results:

• Cryo-EM revealed the structure of TFIIIA and TFIIIC bound to the 5S rRNA promoter, with TFIIIA acting as a DNA-binding adaptor for TFIIIC.
• The study visualized the binding of TFIIIB subunits (Brf1 and TBP) and the subsequent wrapping of the 5S rRNA gene DNA around the complex.
• smFRET demonstrated significant DNA bending and partial dissociation within the complex on a slow timescale.

Conclusions:

• The findings provide unprecedented structural insights into the assembly of the 5S rRNA transcription initiation complex.
• The study elucidates the roles of TFIIIA, TFIIIC, and TFIIIB in promoter recognition and DNA manipulation.
• This work allows for direct comparisons between Pol III and Pol II transcription initiation mechanisms.