Canonical transcription termination mechanisms explain a minority of operons in cyanobacteria

Cyanobacteria are the most abundant phototrophs and hold potential as a carbon-negative platform for bioengineering applications. However, these efforts have been hampered by limited mechanistic understanding of their gene expression, including transcription termination. Unlike most bacteria, cyanobacteria lack the transcription termination factor Rho, raising the speculation that all transcription ends with intrinsic terminators. Here we show that most transcription units (TUs) in Synechococcus elongatus PCC 7942 are not terminated by known termination pathways. Although many TUs (52%) have unique, well-defined 3' ends, only a small fraction have features that resemble canonical intrinsic terminators (30%). The noncanonical 3' ends (22%) broadly lacked strong secondary structure, leaving it unclear how these ends are protected against 3'-5' exonucleolytic decay, but a dinucleotide motif was observed. Furthermore, many TUs (46%) have diverse positions of mRNA 3' ends, suggesting a potentially diffuse termination signal. Finally, we provide evidence that the transcription-repair coupling factor Mfd reduces transcriptional readthrough at the well-defined 3' ends, suggesting that Mfd-dependent termination may be prevalent in this species. Together, our work reveals unique end architectures of the cyanobacterial transcriptome and suggests that undescribed transcription termination mechanisms are active in the phylum. Overall design: Rend-seq mapping of transcription unit architectures including RNA 3' ends at single-nucleotide resolution in the wild-type (WT) freshwater cyanobacterium Synechococcus elongatus PCC 7942 and a mutant deleted for the transcription-repair coupling factor Mfd (Mfd_KO). Rend-seq libraries for two technical replicates of WT (cells collected from the same flask) and two biological replicates of Mfd_KO (separate flasks) were prepared.