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 4 11 (~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.

Region 4 of the RNA polymerase σ subunit counteracts pausing during initial transcription

“…A recent study on initial transcription in vivo (15) and our results suggest that at each nucleotide addition step, ITCs harboring 3-8 nt RNAs can enter into a paused state. The paused ITC (PITC) bifurcates in two pathways: abortive pathway in which nascent RNA dissociates from RNAP, and productive pathway in which nascent RNA remains bound to RNAP and slowly translocates to the next register (Figure 7).…”

“…Nascent RNA chains shorter than 9 nt form unstable hybrids with ssDNA templates and tend to dissociate from the RNAP active site (2,7,11). However, short RNAs (5-6 nt in length) can be stably bound in the paused initially transcribing complex (ITC) formed at the lacUV5 promoter in vitro (12)(13)(14) and in vivo (15). The initial transcription pause occurring after the synthesis of 6-nt RNA functions as a checkpoint on the branched pathway between productive and nonproductive transcription (14).…”

Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences

“…When it occurs, pausing on the pathway to productive initiation delays transcription kinetics on a physiological timescale by affecting promoter escape [ 10 , 11 , 12 , 23 ]. It has been reported that specific sequences in ITS can induce pausing during initiation [ 21 , 24 ]. Therefore, not only the moribund complex, but also RNAP pausing of the productive complex can provide sequence-specific mechanisms controlling transcription during initiation.…”