Nascent RNA profiling reveals genome-wide productive reiterative initiation regulating gene transcription in bacteria

Productive reiterative initiation at promoters is an alternative transcription mechanism characterized by RNA slippage relative to the template DNA and RNA polymerase, resulting in the incorporation of extra nucleotides into transcripts. A comprehensive understanding of the mechanisms underlying productive reiterative initiation, and its functional implications has been hindered due to the complexity of heterogeneous 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. Using this method, we reveal that more than half 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 consecutive 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 stimulate gene transcription. Our results suggest that productive reiterative initiation emerges as an inherent transcription process regulating biological processes independent of protein regulators. Overall design: To identify the genome-wide productive reiterative initiation,5prime terminal native elongating transcript sequencing (5primeTNET-seq) was designed and developed. 5primeTNET-seq was performed using the E. coli strain ß'-WT with His-tag fusion to RpoC (ß') collected at log phase and stationary phase. Nascent RNA in the native elongating ternary complexes pulled down were extracted and size fractioned. The short nascent RNA was purified, treated with or without 5'-monophosphate-dependent terminator exonuclease, and applied to construct library for high-throughput sequencing. RNA-seq was performed using the same strains and conditions to monitor the changes of gene transcription.