Transcription attenuation in synthetic promoters in nonoverlapping tandem formation

Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic
genomes. How their structure and dynamics relate remains unclear,
particularly for tandem formations. To study their transcriptional
interference, we engineered two pairs and one trio of synthetic promoters
in nonoverlapping, tandem formation, in single-copy plasmids transformed
into Escherichia coli cells. From in vivo measurements, we found
that these promoters in tandem formation can have attenuated transcription
rates. The attenuation strength can be widely fine-tuned by the promoters’
positioning, natural regulatory mechanisms, and other factors, including
the antibiotic rifampicin, which is known to hamper RNAP promoter escape.
From this, and supported by in silico models, we concluded that the
attenuation in these constructs emerges from premature terminations
generated by collisions between RNAPs elongating from upstream promoters
and RNAPs occupying downstream promoters. Moreover, we found that these
collisions can cause one or both RNAPs to falloff. Finally, the broad
spectrum of possible, externally regulated, attenuation strengths observed
in our synthetic tandem promoters suggests that they could become useful
as externally controllable regulators of future synthetic circuits.