A sequence‑encoded promoter proximal super pause stabilizes an offline RNA polymerase II state

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Promoter proximal pausing of RNA polymerase II is essential for gene expression regulation in complex organisms.
• The precise roles of DNA sequence and protein factors in mediating this pausing are not fully understood.

Purpose of the Study:

• To develop a novel method for studying transcription regulation with high temporal and parallel resolution.
• To investigate the sequence-specific mechanisms underlying RNA polymerase II pausing.

Main Methods:

• Development of Gene-specific Analysis of Transcriptional Output sequencing (GATO-seq) for reconstituted transcription assays.
• Direct RNA sequencing to map nascent transcript 3' ends from a human gene library.
• Cryogenic-electron microscopy (cryo-EM) to determine high-resolution structures of RNA polymerase II.

Main Results:

• GATO-seq enabled the identification of a potent "super pause" sequence.
• The "super pause" sequence induced pausing resistant to Transcription Factor IIS (TFIIs) rescue.
• Cryo-EM revealed a novel "sidetracked" RNA polymerase II state stabilized by a threonine pocket, limiting backtracking.

Conclusions:

• Nucleic acid sequence directly encodes the propensity for RNA polymerase II pausing.
• Sequence-specific interactions can trap RNA polymerase II in distinct functional states, controlling transcription.
• The developed in vitro technique provides a powerful tool for dissecting transcription regulation.