Abstract
Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites ‘dTSS’, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS ≤ 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.
Highlights
Spaced promoters exist in all branches of life in convergent, divergent, and tandem formations [1,2,3,4,5,6,7]
Tandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs
We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites
Summary
Spaced promoters exist in all branches of life in convergent, divergent, and tandem formations [1,2,3,4,5,6,7]. Models of tandem promoters [8,9,10] have largely been based on measurements of synthetic constructs [11,12,13] and predict that such promoter arrangements result in unique transcription dynamics due to the interference between RNAPs transcribing the promoters [9,10,14,15,16,17,18,19]. Interference will occur when RNAPs elongating from the upstream promoter collide with an RNAP occupying the downstream promoter [14] (in either closed or open complex formation), forcing one of the RNAPs to fall-off (both scenarios are likely possible, and we expect it to differ with, e.g., the binding affinity of the RNAP to the downstream promoter). Even if and when such collisions occur, they are unlikely to result in fall-offs since the RNAPs are moving at similar speeds and in the same direction [9,10,26]
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