XACT-seq comprehensively defines the promoter-position and promoter-sequence determinants for initial-transcription pausing

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 RNAP-active-center position relative to DNA in vivo 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 4-5 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.