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

Author:

Vihervaara AnniinaORCID,Versluis Philip,Himanen Samu V.,Lis John T.ORCID

Abstract

AbstractRNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we develop Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq), which double-selects nascent RNAs and transcription complexes, and track phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncover precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide (+1 nt), through early (+18 to +30 nt) and late (+31 to +60 nt) promoter-proximal pause, and into productive elongation. Pol II CTD is predominantly unphosphorylated from initiation until the early pause-region, whereas serine-2- and serine-5-phosphorylations are preferentially deposited in the later pause-region. Upon pause-release, serine-7-phosphorylation rapidly increases and dominates over the region where Pol II assembles elongation factors and accelerates to its full elongational speed. Interestingly, tracking CTD modifications upon heat-induced transcriptional reprogramming demonstrates that Pol II with phosphorylated CTD remains paused on thousands of heat-repressed genes. These results uncover dynamic Pol II regulation at rate-limiting steps of transcription and provide a nucleotide-resolution technique for tracking composition of engaged transcription complexes.

Publisher

Springer Science and Business Media LLC

Subject

General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary

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