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 (22%). The noncanonical 3' ends broadly lacked strong secondary structure, making it unclear how these ends are protected against 3'-5' exonucleolytic decay. Furthermore, many TUs (46%) have diverse positions of mRNA 3' ends, suggesting a potentially diffuse termination signal. Finally, we observed a moderate increase in RNA levels downstream of most defined 3' ends in the absence of the transcription-repair coupling factor Mfd. This finding indicates that Mfd plays a limited, but widespread, role in RNA end formation, potentially through termination of stalled RNAPs. Together, our work reveals unique end architectures of the cyanobacterial transcriptome and suggests that undescribed transcription termination mechanisms are active in the phylum. ImportanceOur understanding of bacterial transcription regulation is largely based on model organisms like Escherichia coli and Bacillus subtilis, yet many of these mechanisms appear absent or divergent in cyanobacteria. These differences limit our fundamental understanding of gene regulation and the applied potential of cyanobacteria in sustainable biomanufacturing. To address this gap, we characterized transcription termination in the model cyanobacterium Synechococcus elongatus PCC 7942. We resolve a longstanding question by showing that intrinsic termination alone cannot account for most termination events in this organism. Pervasive transcript ends lacking intrinsic terminator features and the absence of Rho suggest the existence of novel termination mechanism(s) and highlight a largely unexplored regulatory landscape. Simultaneously, our work expands the repertoire of functionally characterized cyanobacterial intrinsic terminators, offering a new toolkit to fine-tune gene expression using terminators of defined strengths. These findings pave the way for more predictable and powerful applications of cyanobacteria in green biotechnology.