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.