Nascent elongating transcript sequencing (NET-seq) with SI3 domain-deleted Escherichia coli reveals different pause profiles from wildtype
Transcriptional pausing is a fundamental mechanism that aids gene regulation by cellular RNA polymerases (RNAPs). In many bacterial lineages, a large, surface-exposed domain inserted into the catalytic trigger loop (TL) of RNAP called sequence insertion 3 (SI3) modulates transcription pausing. However, the in vivo roles of SI3 remain largely unknown due in part to the lethality of SI3 deletion. Here we describe construction of a viable Escherichia coli strain with a complete SI3 deletion enabled by a suppressor missense mutation in the portion of rpoC encoding the TL (ß'A941T; ?SI3*). The ?SI3* RNAP exhibited increased transcript elongation rate relative to ?SI3 RNAP lacking the TL substitution, which may explain viability of the ?SI3* strain. Using NET-seq, we found that transcriptional pausing in the ?SI3* strain was increased at some sites and decreased at others compared to wild-type E. coli, except in ribosomal RNA genes. The ?SI3*-enhanced pauses had a sequence motif similar to the consensus elemental pause sequence whereas ?SI3*-suppressed pauses had less similarity to the consensus motif and appeared associated with upstream RNA structures thought to stabilize pausing (pause hairpins; PHs). These putative PH-stabilized pause signals were enriched in 5' untranslated regions and protein coding regions in the E. coli genome. These results suggest that the SI3 domain affects different classes of pause signals differently and imply potential roles of these pauses in protein-coding regions. Overall design: Examination of nascent transcripts in WT and ?SI3* E. coli of biological triplicates. A B. subtilis strain is used as spike-in control for quantitative analysis.