Transcriptional pausing by RNA polymerase has diverse gene regulatory roles; however, the determinants and distribution of these pauses were not well established. The authors of this study sequenced nascent elongating transcripts (NETs) and identified 20,000 new pause sites in known Escherichia coli genes. They defined a 16-nucleotide consensus sequence that is conserved across several bacterial lineages. Investigating the minimal requirements for pausing, the authors found that interactions of RNA polymerase with the DNA template and the transcript that inhibit nucleotide addition were sufficient for pausing in vivo. Finally, the conserved sequence is enriched at translational start sites in both E. coli and Bacillus subtilis, which might cause transcriptional pausing to facilitate RNA folding and ribosome access.