XACT-Seq Comprehensively Defines the Promoter-Position and Promoter-Sequence Determinants for Initial-Transcription Pausing

Jared T. Winkelman, Chirangini Pukhrambam, Irina O. Vvedenskaya, Yuanchao Zhang, Deanne M. Taylor, Premal Shah, Richard H. Ebright, Bryce E. Nickels

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a “stepping” mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a “scrunching” mechanism, has not. We report a method that directly defines the RNAP-active-center position relative to DNA with single-nucleotide resolution (XACT-seq; “crosslink-between-active-center-and-template sequencing”). We apply this method to detect and quantify pausing in initial transcription at 411 (∼4,000,000) promoter sequences in vivo in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4 to 5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard coded by sequence elements similar to those for transcription-elongation pausing.

Original languageEnglish (US)
Pages (from-to)797-811.e8
JournalMolecular cell
Volume79
Issue number5
DOIs
StatePublished - Sep 3 2020

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Keywords

  • RNA polymerase
  • initial transcription
  • massively parallel reporter assay
  • photocrosslinking
  • promoter
  • sigma factor
  • transcription
  • transcription elongation
  • transcription pausing

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