Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner

Area of Science:

• Molecular Biology
• Biophysics

Background:

• RNA polymerase II (RNAP II) transcribes eukaryotic messenger RNAs, a critical and regulated cellular process.
• RNAP II's ability to navigate cellular barriers is key to gene regulation.

Purpose of the Study:

• To investigate the force dynamics of single RNAP II molecules from Saccharomyces cerevisiae.
• To determine the role of the transcription elongation factor TFIIS in RNAP II's response to force and backtracking.

Main Methods:

• Utilized a single-molecule dual-trap optical-tweezers assay.
• Employed a novel method to enrich for active RNAP II complexes.
• Studied RNAP II transcription dynamics in the presence and absence of TFIIS.

Main Results:

• RNAP II backtracking, not just force, dictates transcription limits.
• RNAP II ceased transcription and could not recover from backtracks at ~7.5 pN.
• Backtrack durations followed a t(-3/2) power law, suggesting discrete base-pair diffusion.
• TFIIS rescued backtracked RNAP II, allowing transcription up to ~16.9 pN.

Conclusions:

• Eukaryotic transcription factors like TFIIS can modulate RNAP II's mechanical performance.
• TFIIS enhances RNAP II's ability to overcome backtracking and transcribe against higher forces.
• This highlights a regulatory mechanism for transcription elongation in eukaryotes.