High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Multisubunit RNA polymerases contain a crucial "trigger loop" (TL) essential for transcription.
• The TL plays a multifunctional role in substrate selection, catalysis, and translocation.
• Understanding TL dynamics is key to deciphering transcription mechanisms.

Purpose of the Study:

• To dissect the Saccharomyces cerevisiae RNA polymerase II TL at single-residue resolution.
• To investigate the role of the intra-TL hydrophobic pocket in TL function and catalysis.
• To explore the contribution of funnel and bridge helices (BH) to TL dynamics.

Main Methods:

• Quantitative phenotyping of nearly all TL single variants en masse.
• Analysis of transcription defects and stress responses in mutant classes.
• Assessment of allele-specific genetic interactions between TL and proximal domains.

Main Results:

• Three distinct mutant classes emerged, with specific residue distributions.
• Mutations disrupting the intra-TL hydrophobic pocket led to increased catalysis.
• Genetic interactions supported the role of funnel and bridge helices in TL dynamics.

Conclusions:

• The intra-TL hydrophobic pocket is critical for regulating TL folding and catalytic activity.
• Disruption of this pocket enhances catalytic efficiency.
• The study provides a high-resolution dissection of transcription mechanisms using structural genetics.