PRO-IP-seq tracks molecular modifications of engaged Pol II complexes at nucleotide resolution.

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• Biochemistry

Background:

• RNA Polymerase II (Pol II) is a key enzyme for gene transcription.
• Pol II undergoes dynamic modifications, particularly phosphorylation of its C-terminal domain (CTD), which regulate transcription.
• Understanding these modifications is crucial for deciphering transcription control at rate-limiting steps.

Purpose of the Study:

• To develop a nucleotide-resolution technique for tracking Pol II CTD phosphorylation during transcription.
• To precisely map the positional changes of Pol II CTD phosphorylation as transcription progresses through genes.
• To investigate the role of CTD phosphorylation in transcription pausing and elongation.

Main Methods:

• Development of Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq).
• Simultaneous selection of nascent RNAs and transcription complexes.
• Nucleotide-resolution tracking of Pol II CTD phosphorylation (Ser2, Ser5, Ser7).

Main Results:

• Precise mapping of Pol II CTD phosphorylation from initiation through promoter-proximal pausing and productive elongation.
• Unphosphorylated CTD at initiation, Ser5/Ser2 phosphorylation during pausing, and Ser7 phosphorylation upon pause release.
• Demonstration that phosphorylated Pol II remains paused on heat-repressed genes.

Conclusions:

• CTD phosphorylation is dynamically regulated at nucleotide resolution, controlling key transcription steps.
• PRO-IP-seq provides a powerful tool for studying transcription complex composition and regulation.
• Findings reveal intricate mechanisms of Pol II regulation at rate-limiting steps of transcription.