Nascent RNA profiling reveals regulation of gene transcription through productive reiterative initiation in bacteria

Productive reiterative initiation, an alternative transcription process accompanying RNA slippage relative to the template DNA and RNA polymerase, results in the incorporation of additional nucleotides into transcripts. However, a comprehensive understanding of the mechanisms underlying productive reiterative initiation and its functional implications has been hindered due to the complexity of heterogenous slippage transcripts. Here, we develop and employ 5’ Terminal Native Elongating Transcript Sequencing (5’TNET-seq) to identify and quantify productive reiterative initiation events in Escherichia coli . 5’TNET-seq reveals that over 51% of promoters exhibit productive reiterative initiation. The conserved promoter – 10 region, an appropriate spacer between the –10 region and the TSS, and the transcription initiation region associated with weak RNA-DNA hybrid stability, particularly “AAA” and “TTT” trinucleotide tracts, contribute to high productive reiterative initiation. In addition, up to four nucleotides can be added in a single cycle of productive reiterative initiation. A smaller transcription bubble is observed during productive reiterative initiation, which may stabilize the transcription initiation complex to regulate gene transcription. Our results suggest that productive reiterative initiation emerges as an inherent transcription process regulating biological processes, including cell wall synthesis, independent of protein regulators.