Promoter Architecture Research Articles

Low input capture Hi-C (liCHi-C) identifies promoter-enhancer interactions at high-resolution

Long-range interactions between regulatory elements and promoters are key in gene transcriptional control; however, their study requires large amounts of starting material, which is not compatible with clinical scenarios nor the study of rare cell populations. Here we introduce low input capture Hi-C (liCHi-C) as a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. As proof of its broad applicability, we implement liCHi-C to study normal and malignant human hematopoietic hierarchy in clinical samples. We demonstrate that the dynamic promoter architecture identifies developmental trajectories and orchestrates transcriptional transitions during cell-state commitment. Moreover, liCHi-C enables the identification of disease-relevant cell types, genes and pathways potentially deregulated by non-coding alterations at distal regulatory elements. Finally, we show that liCHi-C can be harnessed to uncover genome-wide structural variants, resolve their breakpoints and infer their pathogenic effects. Collectively, our optimized liCHi-C method expands the study of 3D chromatin organization to unique, low-abundance cell populations, and offers an opportunity to uncover factors and regulatory networks involved in disease pathogenesis.

P317: Gene therapy base editors reshape single-molecule chromatin architecture of γ-globin promoters

P317: Gene therapy base editors reshape single-molecule chromatin architecture of γ-globin promoters

Regulatory Mechanisms for Transcriptional Bursting Revealed by an Event-Based Model.

Gene transcription often occurs in discrete bursts, and it can be difficult to deduce the underlying regulatory mechanisms for transcriptional bursting with limited experimental data. Here, we categorize numerous states of single eukaryotic genes and identify 6 essential transcriptional events, each comprising a series of state transitions; transcriptional bursting is characterized as a sequence of 4 events, capable of being organized in various configurations, in addition to the beginning and ending events. By associating transcriptional kinetics with mean durations and recurrence probabilities of the events, we unravel how transcriptional bursting is modulated by various regulators including transcription factors. Through analytical derivation and numerical simulation, this study reveals key state transitions contributing to transcriptional sensitivity and specificity, typical characteristics of burst profiles, global constraints on intrinsic transcriptional noise, major regulatory modes in individual genes and across the genome, and requirements for fast gene induction upon stimulation. It is illustrated how biochemical reactions on different time scales are modulated to separately shape the durations and ordering of the events. Our results suggest that transcriptional patterns are essentially controlled by a shared set of transcriptional events occurring under specific promoter architectures and regulatory modes, the number of which is actually limited.

Genome-wide inference reveals that feedback regulations constrain promoter-dependent transcriptional burst kinetics.

Gene expression in mammalian cells is highly variable and episodic, resulting in a series of discontinuous bursts of mRNAs. A challenge is to understand how static promoter architecture and dynamic feedback regulations dictate bursting on a genome-wide scale. Although single-cell RNA sequencing (scRNA-seq) provides an opportunity to address this challenge, effective analytical methods are scarce. We developed an interpretable and scalable inference framework, which combined experimental data with a mechanistic model to infer transcriptional burst kinetics (sizes and frequencies) and feedback regulations. Applying this framework to scRNA-seq data generated from embryonic mouse fibroblast cells, we found Simpson's paradoxes, i.e. genome-wide burst kinetics exhibit different characteristics in two cases without and with distinguishing feedback regulations. We also showed that feedbacks differently modulate burst frequencies and sizes and conceal the effects of transcription start site distributions on burst kinetics. Notably, only in the presence of positive feedback, TATA genes are expressed with high burst frequencies and enhancer-promoter interactions mainly modulate burst frequencies. The developed inference method provided a flexible and efficient way to investigate transcriptional burst kinetics and the obtained results would be helpful for understanding cell development and fate decision.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Promoter sequence and architecture determine expression variability and confer robustness to genetic variants.

Genetic and environmental exposures cause variability in gene expression. Although most genes are affected in a population, their effect sizes vary greatly, indicating the existence of regulatory mechanisms that could amplify or attenuate expression variability. Here, we investigate the relationship between the sequence and transcription start site architectures of promoters and their expression variability across human individuals. We find that expression variability can be largely explained by a promoter's DNA sequence and its binding sites for specific transcription factors. We show that promoter expression variability reflects the biological process of a gene, demonstrating a selective trade-off between stability for metabolic genes and plasticity for responsive genes and those involved in signaling. Promoters with a rigid transcription start site architecture are more prone to have variable expression and to be associated with genetic variants with large effect sizes, while a flexible usage of transcription start sites within a promoter attenuates expression variability and limits genotypic effects. Our work provides insights into the variable nature of responsive genes and reveals a novel mechanism for supplying transcriptional and mutational robustness to essential genes through multiple transcription start site regions within a promoter.

Structural basis of three different transcription activation strategies adopted by a single regulator SoxS.

Transcription activation is established through extensive protein-protein and protein-DNA interactions that allow an activator to engage and remodel RNA polymerase. SoxS, a global transcription activator, diversely regulates subsets of stress response genes with different promoters, but the detailed SoxS-dependent transcription initiation mechanisms remain obscure. Here, we report cryo-EM structures of three SoxS-dependent transcription activation complexes (SoxS-TACI, SoxS-TACIIand SoxS-TACIII) comprising of Escherichia coli RNA polymerase (RNAP), SoxS proteinand three representative classes of SoxS-regulated promoters. The structures reveal that SoxS monomer orchestrates transcription initiation through specific interactions with the promoter DNA and different conserved domains of RNAP. In particular, SoxS is positioned in the opposite orientation in SoxS-TACIII to that in SoxS-TACI and SoxS-TACII, unveiling a novel mode of transcription activation. Strikingly, two universally conserved C-terminal domains of alpha subunit (αCTD) of RNAP associate with each other, bridging SoxS and region 4 of σ70. We show that SoxS interacts with RNAP directly and independently from DNA, remodeling the enzyme to activate transcription from cognate SoxS promoters while repressing transcription from UP-element containing promoters. Our data provide a comprehensive summary of SoxS-dependent promoter architectures and offer new insights into the αCTD contribution to transcription control in bacteria.

Saccharomyces cerevisiae cis-acting DNA sequences curation pipeline (Sc-cADSs-CP): Master transcription factors prediction in yeasts

Designing promoter architectures hinges on genomic and functional annotation. Saccharomyces cerevisiae is the first model yeast whose databases host genomic and functional annotation information. To predict transcription factors (TFs) regulating central pathways in yeasts, we first introduce S. cerevisiae cis-acting DNA sequences/sites (cADSs) curation pipeline (Sc-cADSs-CP). The promoters of the genes involved in the central pathways of S. cerevisiae were retrieved from the genome sequences. We processed the binding frequency matrices of TFs with the following two criteria. First, we extracted cADSs based on the TF motifs in the TRANSFAC database; then, if there were more than one frequency matrix for a TF, the longest one with the maximum sensitivity was used. Next, we developed the direct scanning algorithm ScanAlgo (uses the Biopython-library), scanning DNA motifs for pairing cross-species alignments. We used the tools Sc-cADSs-CP and ScanAlgo to predict master TFs for Pichia pastoris, which lacks extensive functional annotation studies. The phylogenetic footprinting results were obtained by aligning the scanned S. cerevisiae promoters against orthologous P. pastoris promoters. The predicted cADSs were summed into position weight matrices unique to P. pastoris. We annotated 116 TFs based on the phylogenetic footprinting predictions of cADSs regulating the central pathways in P. pastoris. The presented methodology with the tools Sc-cADSs-CP and ScanAlgo enables the prediction of master TFs and cADSs in yeasts.

Promoter architecture of Drosophila genes regulated by Myocyte enhancer factor-2.

To gain understanding into the mechanisms of transcriptional activation of muscle genes, we sought to determine if genes targeted by the myogenic transcription factor Myocyte enhancer factor-2 (MEF2) were enriched for specific core promoter elements. We identified 330 known MEF2 target promoters in Drosophila, and analyzed them for for the presence and location of 17 known consensus promoter sequences. As a control, we also searched all Drosophila RNA polymerase II-dependent promoters for the same sequences. We found that promoter motifs were readily detected in the MEF2 target dataset, and that many of them were slightly enriched in frequency compared to the control dataset. A prominent sequence over-represented in the MEF2 target genes was NDM2, that appeared in over 50% of MEF2 target genes and was 2.5-fold over-represented in MEF2 targets compared to background. To test the functional significance of NDM2, we identified two promoters containing a single copy of NDM2 plus an upstream MEF2 site, and tested the activity of these promoters in vivo. Both the sticks and stones and Kahuli fragments showed strong skeletal myoblast-specific expression of a lacZ reporter in embryos. However, the timing and level of reporter expression was unaffected when the NDM2 site in either element was mutated. These studies identify variations in promoter architecture for a set of regulated genes compared to all RNA polymerase II-dependent genes, and underline the potential redundancy in the activities of some core promoter elements.

Reliance of Host-Encoded Regulators of Retromobility on Ty1 Promoter Activity or Architecture.

The Ty1 retrotransposon family is maintained in a functional but dormant state by its host, Saccharomyces cerevisiae. Several hundred RHF and RTT genes encoding co-factors and restrictors of Ty1 retromobility, respectively, have been identified. Well-characterized examples include MED3 and MED15, encoding subunits of the Mediator transcriptional co-activator complex; control of retromobility by Med3 and Med15 requires the Ty1 promoter in the U3 region of the long terminal repeat. To characterize the U3-dependence of other Ty1 regulators, we screened a library of 188 known rhf and rtt mutants for altered retromobility of Ty1his3AI expressed from the strong, TATA-less TEF1 promoter or the weak, TATA-containing U3 promoter. Two classes of genes, each including both RHFs and RTTs, were identified. The first class comprising 82 genes that regulated Ty1his3AI retromobility independently of U3 is enriched for RHF genes that restrict the G1 phase of the cell cycle and those involved in transcriptional elongation and mRNA catabolism. The second class of 51 genes regulated retromobility of Ty1his3AI driven only from the U3 promoter. Nineteen U3-dependent regulators (U3DRs) also controlled retromobility of Ty1his3AI driven by the weak, TATA-less PSP2 promoter, suggesting reliance on the low activity of U3. Thirty-one U3DRs failed to modulate P PSP2 -Ty1his3AI retromobility, suggesting dependence on the architecture of U3. To further investigate the U3-dependency of Ty1 regulators, we developed a novel fluorescence-based assay to monitor expression of p22-Gag, a restriction factor expressed from the internal Ty1i promoter. Many U3DRs had minimal effects on levels of Ty1 RNA, Ty1i RNA or p22-Gag. These findings uncover a role for the Ty1 promoter in integrating signals from diverse host factors to modulate Ty1 RNA biogenesis or fate.

Mitigating Host Burden of Genetic Circuits by Engineering Autonegatively Regulated Parts and Improving Functional Prediction.

Mitigating unintended interferences between circuits and host cells is key to realize applications of synthetic regulatory systems both for bacteria and mammalian cells. Here, we demonstrated that growth burden and circuit dysregulation occurred in a concentration-dependent manner for specific transcription factors (CymR*/CymR) in E.coli, and direct negative feedback modules were able to control the concentration of CymR*/CymR, mitigate growth burden, and restore circuit functions. A quantitative design scheme was developed for circuits embedded with autorepression modules. Four key parameters were theoretically identified to determine the performance of autoregulated switches and were experimentally modified by fine-tuning promoter architectures and cooperativity. Using this strategy, we synthesized a number of switches and demonstrated its improvement of product titers and host growth controlling the complex deoxyviolacein biosynthesis pathway. Furthermore, we restored functions of a dysregulated multilayer NOR gate by integrating autorepression modules. Our work provides a blueprint for engineering host-adaptable synthetic systems.

Genome-wide analysis of cowpea NAC transcription factor family elucidating the genetic & molecular relationships that interface stress and growth regulatory signals

Being a key transcriptional mediator of stress and growth responses, NAC transcription factors hold the paramount potential to develop climate-smart pulse crops. Despite their seminal role, they are yet unexplored in many important orphan legumes like cowpea. The recent availability of a comprehensive reference genome motivated us to investigate and assess the functional importance of the remarkable NAC family in cowpea for sustainable crop research. This study identified 130 NAC proteins in cowpea, namely VuNAC01-130, classified into 8 phylogenetic groups. 27 cowpea-specific members were clustered as a distinct clade with no close orthologs from other species, implicating novel functions. VuNAC proteins carried multipartite nuclear signals and unique transactivation regions with conserved patterns. 18 proteins were associated with non-NAC chimeric domains. The genes owned a unique promoter architecture encompassing pyrimidine-rich elements. The family manifested prominent segmental and tandem chromosomal duplication resulting in numerous stress-responsive members and large paralogous groups. The promoter and interactome analysis revealed multi-tier regulation through light, hormone, and transcription factors (NAC/MYB/WRKY/ERF and Dof/TCP), suggesting a cross-talk between stress and growth-regulating signals. Besides, the TFs were associated with metabolic processes such as trehalose and folate synthesis, carbohydrate transport, lipid signaling, and electron transfer. Comparatively, ATAF-like members (Group Ia) would be the most promising candidates to develop climate-smart crops with improved stress adaptation and agronomic traits by translational approach, due to their predicted functional versatility.

A functional promoter from the archaeon Halobacterium salinarum is also transcriptionally active in E. coli

BackgroundArchaea form a third domain of life that is distinct from Bacteria and Eukarya. So far, many scholars have elucidated considerable details about the typical promoter architectures of the three domains of life. However, a functional promoter from the archaeon Halobacterium salinarum has never been studied in Escherichia coli.ResultsThis paper found that the promoter of Halobacterium salinarum showed a promoter function in Escherichia coli. This Escherichia coli promoter structure contains − 10 box, -10 box extension and − 29 elements, however, no -35 box. The − 29 element is exercised by the TATA box in archaea. And we isolated the RM10 fragment that possessed the fusion characteristics of bacteria and archaea, which was overlapped with functionality of TATA box and − 29 elements.ConclusionsThe − 29 element reflects the evolutionary relationship between the archaeal promoter and the bacterial promoter. The result possibly indicated that there may be a certain internal connection between archaea and bacteria. We hypothesized that it provided a new viewpoint of the evolutionary relationship of archaea and other organisms.

Delineation of the DNA Structural Features of Eukaryotic Core Promoter Classes.

The eukaryotic transcription is orchestrated from a chunk of the DNA region stated as the core promoter. Multifarious and punctilious core promoter signals, viz., TATA-box, Inr, BREs, and Pause Button, are associated with a subset of genes and regulate their spatiotemporal expression. However, the core promoter architecture linked with these signals has not been investigated exhaustively for several species. In this study, we attempted to envisage the adaptive binding landscape of the transcription initiation machinery as a function of DNA structure. To this end, we deployed a set of k-mer based DNA structural estimates and regular expression models derived from experiments, molecular dynamic simulations, and theoretical frameworks, and high-throughout promoter data sets retrieved from the eukaryotic promoter database. We categorized protein-coding gene core promoters based on characteristic motifs at precise locations and analyzed the B-DNA structural properties and non-B-DNA structural motifs for 15 different eukaryotic genomes. We observed that Inr, BREd, and no-motif classes display common patterns of DNA sequence and structural environment. TATA-containing, BREu, and Pause Button classes show a deviant behavior with the TATA class displaying varied axial and twisting flexibility while BREu and Pause Button leaned toward G-quadruplex motif enrichment. Intriguingly, DNA meltability and shape signals are conserved irrespective of the presence or absence of distinct core promoter motifs in the majority of species. Altogether, here we delineated the conserved DNA structural signals associated with several promoter classes that may contribute to the chromatin configuration, orchestration of transcription machinery, and DNA duplex melting during the transcription process.

Genetic background and cis-organization regulate ALDH7B4 gene expression in Eutrema salsugineum: a promoter analysis case study.

ALDH7B4 promoter analysis in A. thaliana and E. salsugineum reveals that both genetic background and promoter architecture contribute to gene expression in response to stress in different species. Many genes are differentially regulated in a comparison of salinity-sensitive and salinity-tolerant plant species. The aldehyde dehydrogenase 7B4 (ALDH7B4) gene is turgor-responsive in A. thaliana and encodes a highly conserved detoxification enzyme in plants. This study compared the ALDH7B4 gene in A. thaliana (salinity-sensitive) and in the salinity-tolerant close relative Eutrema salsugineum. EsALDH7B4 in E.salsugineum is the ortholog of AtALDH7B4 and the expression is also salinity, drought, and wound responsive. However, E. salsugineum requires higher salinity stress to induce the EsALDH7B4 transcriptional response. The GUS expression driven either by the promoter AtALDH7B4 or EsALDH7B4 was induced under 300mM NaCl treatment in A. thaliana while 600mM NaCl treatment was required in E. salsugineum, suggesting that the genetic background plays a crucial role in regulation of gene expression. Promoter sequences of ALDH7B4 are less conserved than the protein coding region. A series of EsALDH7B4 promoter deletion fragments were fused to the GUS reporter gene and promoter activity was determined in A. thaliana. The promoter region that contains two conserved ACGT-containing motifs was identified to be essential for stress induction. Furthermore, a 38bp "TC" rich motif in the EsALDH7B4 promoter, absent from the AtALDH7B4 promoter, negatively affects EsALDH7B4 expression. A MYB-like transcription factor was identified to bind the "TC" motif and to repress the EsALDH7B4 promoter activity. This study reveals that genetic background and cis-acting elements coordinately regulate gene expression.

Exact distributions for stochastic gene expression models with arbitrary promoter architecture and translational bursting.

Gene expression in individual cells is inherently variable and sporadic, leading to cell-to-cell variability in mRNA and protein levels. Recent single-cell and single-molecule experiments indicate that promoter architecture and translational bursting play significant roles in controlling gene expression noise and generating the phenotypic diversity that life exhibits. To quantitatively understand the impact of these factors, it is essential to construct an accurate mathematical description of stochastic gene expression and find the exact analytical results, which is a formidable task. Here, we develop a stochastic model of bursty gene expression, which considers the complex promoter architecture governing the variability in mRNA expression and a general distribution characterizing translational burst. We derive the analytical expression for the corresponding protein steady-state distribution and all moment statistics of protein counts. We show that the total protein noise can be decomposed into three parts: the low-copy noise of protein due to probabilistic individual birth and death events, the noise due to stochastic switching between promoter states, and the noise resulting from translational busting. The theoretical results derived provide quantitative insights into the biochemical mechanisms of stochastic gene expression.

Genomic architecture of promoters and transcriptional regulation of candidate genes in rice involved in tolerance to anaerobic germination

Global climate change has significant impacts on rice production due to flooding caused by heavy rainfall which has serious implications in rice growing areas of Southeast Asia. In these areas, direct seeding is preferred as it saves labour, cost, energy and water. Hence, the unique ability of rice seeds to germinate under anaerobic condition is important to achieve direct seeding. A number of candidate genes associated with anaerobic germination have been identified through genome-wide association studies (GWAS) based on single nucleotide polymorphism (SNP) markers. These candidate genes could be regulated by unique transcriptional regulatory mechanisms that enable them to tolerate anaerobic stress. However, the molecular basis of this transcriptional regulation is essentially unknown. Hence, a comprehensive understanding of the gene regulation of these candidate genes can provide important insights towards improved rice varieties for direct seeding. To fill this gap, publicly available novel candidate genes [Os01g0911700 (OsVP1), Os05g0560900 (OsGA2ox8), Os05g0562200 (OsDi19–1) and Os06g0548200] associated with anaerobic germination tolerance were selected and analysed. The genomic architecture of the promoters of these genes was defined by performing putative cis-elements enrichment analysis. Promoter analysis identified putative cis-elements linked to transcription factors (TFs) such as MYB, bZIP, AP2/ERF, EIN3, ABI3, ABR1, ARF, WRKY, bHLH, BES1/BZR1, ZnF, NAC, DOF, AS2, HSF, HD-Zip, TCP, MADS-box, ARR-B, E2F and GATA. Interestingly, putative cis-elements associated with unique TFs possibly related to epigenetic effect such as MYB3R, TSO1, TCX2/SOL2, TCX3/SOL1 and ASHR1; regulation of lipid metabolism associated with AHL and, regulation of sugar and fatty acid metabolism associated with WRI1 were also identified. Besides, the analysis also provides useful information on the crosstalk between key TFs and phytohormones such as GA, ABA, ethylene, auxin, JA and BR for unique transcriptional regulation leading to coleoptile elongation of rice during anaerobic germination tolerance. The current in silico analysis can provide information and contribute to the development of new rice varieties for direct seeding.

Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages

RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5′ and 3′ boundaries of transcripts, confounding the discovery of key transcription initiation and termination events as well as operon architectures. Yet, the elucidation of these elements is crucial for the understanding of the strategy of transcription regulation during the infection process, which is currently lacking beyond a handful of model phages. We developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study transcription of Pseudomonas aeruginosa phage LUZ7, obtaining a comprehensive genome-wide map of viral transcription start sites, terminators, and complex operon structures that fine-regulate gene expression. Our work provides new insights in the RNA biology of a non-model phage, unveiling distinct promoter architectures, putative small non-coding viral RNAs, and the prominent regulatory role of terminators during infection. The robust workflow presented here offers a framework to obtain a global, yet fine-grained view of phage transcription and paves the way for standardized, in-depth transcription studies for microbial viruses or bacteria in general.

Nucleo-cytoplasmic interactions affect the 5’ terminal transcription of mitochondrial genes between the isonuclear CMS line UG93A and its maintainer line UG93B of kenaf (Hibiscus cannabinus)

Gene expression and translation in plant mitochondria remain poorly understood due to the complicated transcription of its mRNA. In this study, we report the 5' and 3' RNA extremities and promoters of five mitochondrial genes, atp1, atp4, atp6, atp9, and cox3. The results reveal that four genes (atp1, atp4, atp6, and cox3) are transcribed from multiple initiation sites but with a uniform transcript at the 3' end, indicating that heterogeneity of the 5' end is a common feature in the transcription of kenaf mitochondrial genes. Furthermore, we found that the transcription initiation sites of these four genes are significantly different in UG93A, UG93B, and the F1 hybrid. These data indicate that nuclear loci and unknown transcription factors within the mitochondria of different cytoplasmic types may be involved in mitochondrial transcription. Promoter architecture analysis showed that the promoter core sequences are conserved in the kenaf mitochondrial genome but are highly divergent, suggesting that these elements are essential for the promoter activity of mitochondrial genes in kenaf. Our results reveal that the heterogeneity of the 5' end and uniformity at the 3' end are common transcriptional features of mitochondrial genes. These data provide essential information for understanding the transcription of mitochondrial genes in kenaf and can be used as a reference for other plants.

The bacterial promoter spacer modulates promoter strength and timing by length, TG-motifs and DNA supercoiling sensitivity

Transcription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals an extension to the -10 region that enhances RNAP binding but damps promoter activity. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.