Analytical kinetic model of native tandem promoters in E. coli
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA759250 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15680311_1.fastq.gz | Fastqsanger.gz | |||
| SRR15680311_2.fastq.gz | Fastqsanger.gz | |||
| SRR15680312_1.fastq.gz | Fastqsanger.gz | |||
| SRR15680312_2.fastq.gz | Fastqsanger.gz | |||
| SRR15680313_1.fastq.gz | Fastqsanger.gz |
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Project description:Tandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. 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. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.
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Project description: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.
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Project description:The core promoter is the regulatory sequence to which RNA polymerase is recruited and where it acts to initiate transcription. Here, we present the first comprehensive study of yeast core promoters, providing massively parallel measurements of core promoter activity and of TSS locations and relative usage for thousands of native and designed sequences. We found core promoter activity to be highly correlated to the activity of the entire promoter, and that sequence variation in different core promoter regions substantially tunes its activity in a predictable way. We also show that location, orientation and flanking bases critically affect TATA element function, that transcription initiation in highly active core promoters is focused within a narrow region, that poly(dA:dT) orientation has functional consequence at the 3' end of promoters, and that orthologous core promoters across yeast species have conserved activities. Our results demonstrate the importance of core promoters in the quantitative study of gene regulation.
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Project description:CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCF-binding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in vivo in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.
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Project description:CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCF-binding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in vivo in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.
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Project description:CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCF-binding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in vivo in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.
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