Promoter-independent gene activation by Sigma factor-associated Cas12f proteins
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PROVIDER: PRJNA1247282 | ENA |
REPOSITORIES: ENA
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Project description:Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP). Although the 7 σ factors in E. coli have been extensively characterized, Bacteroidetes species encode dozens of specialized σ factors whose precise functions are unknown, suggesting additional layers of regulatory potential. Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σE factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σE homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a surprisingly minimal 5ʹ-G target-adjacent motif (TAM). Recruitment of σE was dependent on dCas12f and gRNA, suggesting a direct protein-protein complex, and co-expression experiments demonstrated that the ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σE complexes drove potent gene expression in the absence of any requisite promoter motifs, with transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided gene activation that embodies natural features reminiscent of CRISPRa technology developed by humans.
2025-07-30 | GSE293889 | GEO
Project description:The ability of bacteria to adapt to stress depends on their ability to conditionally express specific sets of genes. Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma (σ) factors that regulate functions important for survival under conditions eliciting cell envelope stress. Of these, four have been studied in detail: σM, σW, σX and σV . The regulons of these four σ factors overlap, although the sequences that determine promoter selectivity are incompletely understood. A major role in promoter selectivity has been ascribed to the core promoter -10 and -35recognition elements. Here, we demonstrate that a homopolymeric T-tract motif, proximal to the-35 recognition element, functions in combination with the core promoter sequences to2determine selectivity for ECF sigma factors. This motif is critical for promoter activation by σV, contributes to activation by σM and σX, but has relatively little effect for σW. We propose that this motif, which is a feature of the deduced promoter consensus for a subset of ECF s factors from many species, affects confirmation or curvature of the DNA to influence promoter activity. The differential effect of this region amongst ECF s factors thereby provides a mechanism to modulate the nature and extent of regulon overlap.
2017-02-28 | GSE95393 | GEO
Project description:Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σ(A), by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σ(A) is -10 specific. However, the promoter -10 specific interaction is unable to allow the σ(A) to discern the optimal promoter spacing. To fulfill this goal, the σ(A) requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function.
| S-EPMC3035472 | biostudies-literature
Project description:Abstract of associated manuscript: The Bacillus subtilis extracytoplasmic function (ECF) sigma(M) factor is activated by cell envelope stress elicited by antibiotics, and by acid, heat, ethanol and superoxide stresses. Here, we have used several complementary approaches to identify genes controlled by sigma(M). In many cases, expression is only partially dependent on sigma(M) because of both overlapping promoter recognition with other ECF sigma factors and the presence of additional promoter elements. Genes regulated by sigma(M) have a characteristic pattern of induction in response to cell envelope-acting antibiotics as evidenced by hierarchical clustering analysis. sigma(M) also contributes to the expression of the Spx transcription factor and thereby indirectly regulates genes of the Spx regulon. Cell envelope stress responses also include regulons controlled by sigma(W), sigma(B) and several two-component regulatory systems (e.g. LiaRS, YycFG, BceRS). Activation of the sigma(M) regulon increases expression of proteins functioning in transcriptional control, cell wall synthesis and shape determination, cell division, DNA damage monitoring, recombinational repair and detoxification.
2008-12-05 | GSE13820 | GEO
Project description:Characterization of S.lividans TK24 secreted proteome changes upon deletion of certain sigma factors.
2019-02-16 | PXD006818 | Pride
Project description:An alternative sigma factor (σ32) recognizes the unique set of promoters upon heat shock. Here, we determined 54 σ32 promoters at nucleotide resolution using ChIP-exo, enabling us to compare those with housekeeping σ70 promoters. The results elucidated the overarching principles of promoter overlapping between the two σ-factors, which are sequence-specific non-, half-, and full-shared modes with a perfect sequence conservativeness of −35 element as a key determinant of full-shared mode.
2020-08-12 | GSE60668 | GEO
Project description:An alternative sigma factor (σ32) recognizes the unique set of promoters upon heat shock. Here, we determined 54 σ32 promoters at nucleotide resolution using ChIP-exo, enabling us to compare those with housekeeping σ70 promoters. The results elucidated the overarching principles of promoter overlapping between the two σ-factors, which are sequence-specific non-, half-, and full-shared modes with a perfect sequence conservativeness of −35 element as a key determinant of full-shared mode.
2020-08-12 | GSE60667 | GEO
Project description:Escherichia coli uses σ factors to quickly control large gene cohorts during stress conditions. While most of its genes respond to a single σ factor, approximately 5% of them have dual σ factor preference. The most common are those responsive to both σ70, which controls housekeeping genes, and σ38, which activates genes during stationary growth and stresses. Using RNA-seq and flow-cytometry measurements, we show that ‘σ70+38 genes’ are nearly as upregulated in stationary growth as ‘σ38 genes’. Moreover, we find a clear quantitative relationship between their promoter sequence and their response strength to changes in σ38 levels. We then propose and validate a sequence dependent model of σ70+38 genes, with dual sensitivity to σ38 and σ70, that is applicable in the exponential and stationary growth phases, as well in the transient period in between. We further propose a general model, applicable to other stresses and σ factor combinations. Given this, promoters controlling σ70+38 genes (and variants) could become important building blocks of synthetic circuits with predictable, sequence-dependent sensitivity to transitions between the exponential and stationary growth phases.
2022-03-22 | GSE188752 | GEO
Project description:To engineer synthetic gene circuits, molecular building blocks are developed which can modulate gene expression without interference, mutually or with the host’s cell machinery. Promoter libraries of E. coli sigma factor 70 and B. subtilis B-, F- and W-dependent promoters are exploited to construct prediction models, capable of both predicting promoter TIF and orthogonality of the specific promoters. This is achieved by the creation of high-throughput DNA sequencing data from fluorescence-activated cell sorted promoter libraries.
2020-08-01 | E-MTAB-8734 | biostudies-arrayexpress
Project description:When performed at single bp resolution, the genome-wide location, occupancy level, and structural organization of DNA binding proteins provides mechanistic insights into genome regulation. Here we use ChIP-exo to provide the first high resolution view of the epigenomic organization of the E. coli transcription machinery and nucleoid structural proteins when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We suggest that indirect readout of DNA shape at the flanks of cognate motifs provide major contributions to site specificity at promoter positions -35/-24 and -10/-12. We examined the site specificity of three sigma factors (RpoD/70, RpoH/32, and RpoN/54), RNA polymerase (RNAP or RpoA, B, C) and two nucleoid proteins (Fis and IHF). Our results confirm and refine reports that RpoD binds most annotated promoters, whereas RpoH and RpoN bind a much smaller subset, each through their cognate motifs. However, only upon heat shock does RpoH becomes active for RNAP recruitment. In contrast, upon heat shock RpoD remains active at its cognate promoters (including at heat shock genes), whereas RpoN remains inactive at its cognate promoters. RpoN binds ~1,000 non-annotated RpoN motifs, which may reflect a large number of condition-specific transcription units. Occupancy patterns of sigma factors and RNAP suggest a common promoter recruitment mechanism that differs from the long-standing views that sigma and RNAP are co-recruited as a complex, and also simultaneously dissociate from promoters. Our findings suggest that sigma factors are recruited and/or maintained at most promoters via an RNAP-independent mechanism. When RNAP arrives, it dwells for a relatively short time before clearing the promoter, leaving sigma behind. Taken together these findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape and redefine mechanistic steps in regulated promoter assembly in E. coli.
2022-02-21 | GSE189206 | GEO