Gene expression in bacteria is organized into operons, functional units of genomic DNA containing clusters of genes under the control of single promoters. The definition of an operon relies on the precise identification of the start of transcription (TSS), the end of transcription and (TTS) and the relation between the TSS and the TTS for each transcript molecule. Yet current experimental methods for sequencing RNA in bacteria rely on the necessary fragmentation of transcripts for short read sequencing. Consequently the larger context by which genes are expressed and the phasing between the start and the end of primary transcripts are lost. As a result, and despite overwhelming mass of transcriptome information, accurate operon structure have so far been solved only for a handful of cases. Here we describe SMRT‐cappable‐seq as the first experimental methodology to identify operons genome‐wide in prokaryotes. It combines the isolation of full‐length primary transcripts with long read SMRT sequencing. Applied to E.coli, SMRT‐cappable‐seq identifies a total of 2300 operons from which around 900 are novel. Importantly, our result reveals a pervasive read‐through of previous experimentally validated transcription termination sites. Termination read‐through represents a powerful strategy to control gene expression and redefines operons with thousand of new operons containing more genes than previously thought. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents an invaluable resource for understanding gene regulation and function in bacteria.Support or Funding InformationNew England Biolabs Inc. and Pacific Biosciences of California, Inc supported this research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.