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

Pausing by RNA polymerase 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 RNA polymerase 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 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.