Supercoiling Sensitivity Research Articles

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.

Role of the Discriminator Sequence in the Supercoiling Sensitivity of Bacterial Promoters.

ABSTRACTDNA supercoiling acts as a global transcriptional regulator that contributes to the rapid transcriptional response of bacteria to many environmental changes. Although a large fraction of promoters from phylogenetically distant species respond to superhelical variations, the sequence or structural determinants of this behavior remain elusive. Here, we focus on the sequence of the “discriminator” element that was shown to modulate this response in several promoters. We develop a quantitative thermodynamic model of this regulatory effect, focusing on open complex formation during transcription initiation independently from promoter-specific regulatory proteins. We analyze previous and new expression data and show that the model predictions quantitatively match the in vitro and in vivo supercoiling response of selected promoters with mutated discriminator sequences. We then test the universality of this mechanism by a statistical analysis of promoter sequences from transcriptomes of phylogenetically distant bacteria under conditions of supercoiling variations (i) by gyrase inhibitors, (ii) by environmental stresses, or (iii) inherited in the longest-running evolution experiment. In all cases, we identify a robust and significant sequence signature in the discriminator region, suggesting that supercoiling-modulated promoter opening underpins a ubiquitous regulatory mechanism in the prokaryotic kingdom based on the fundamental mechanical properties of DNA and its basal interaction with RNA polymerase.IMPORTANCE In this study, we highlight the role of the discriminator as a global sensor of supercoiling variations and propose the first quantitative regulatory model of this principle, based on the specific step of promoter opening during transcription initiation. It defines the predictive rule by which DNA supercoiling quantitatively modulates the expression rate of bacterial promoters, depending on the G/C content of their discriminator and independently from promoter-specific regulatory proteins. This basal mechanism affects a wide range of species, which is tested by an extensive analysis of global high-throughput expression data. Altogether, ours results confirm and provide a quantitative framework for the long-proposed notion that the discriminator sequence is a significant determinant of promoter supercoiling sensitivity, underpinning the ubiquitous regulatory action of DNA supercoiling on the core transcriptional machinery, in particular in response to quick environmental changes.

Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling.

DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes’ local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.

Transcription-coupled DNA supercoiling dictates the chromosomal arrangement of bacterial genes.

Over the recent decade, the central importance of DNA supercoiling in chromosome organization and global gene regulation of bacteria became more and more visible. With a regulon comprising more than 2000 genes in Escherichia coli, DNA supercoiling is among the most influential regulators of gene expression found in bacteria so far. However, the mechanism creating thousands of diverse temporal gene expression patterns coordinated by DNA supercoiling remains unclear. In this study we show that a specific chromosomal arrangement of genes modulates the local levels of DNA supercoiling at gene promoters via transcription-coupled DNA supercoiling (TCDS) in the model organism E. coli. Our findings provide a consistent explanation for the strong positive coupling of temporal gene expression patterns of neighboring genes. Using comparative genomics we are furthermore able to provide evidence that TCDS is a driving force for the evolution of chromosomal gene arrangement patterns in other Enterobacteriaceae. With the currently available data of promoter supercoiling sensitivity we prove that the same principle is applicable also for the evolutionary distant gram-positive pathogenic bacterium Streptococcus pneumoniae. Moreover, our findings are fully consistent with recent investigations concerning the regulatory impact of TCDS on gene pairs in eukaryots underpinning the broad applicability of our analysis.

Structural attributes of nucleotide sequences in promoter regions of supercoiling-sensitive genes: How to relate microarray expression data with genomic sequences

The level of supercoiling in the chromosome can affect gene expression. To clarify the basis of supercoiling sensitivity, we analyzed the structural features of nucleotide sequences in the vicinity of promoters for the genes with expression enhanced and decreased in response to loss of chromosomal supercoiling in Escherichia coli. Fourier analysis of promoter sequences for supercoiling-sensitive genes reveals the tendency in selection of sequences with helical periodicities close to 10nt for relaxation-induced genes and to 11nt for relaxation-repressed genes. The helical periodicities in the subsets of promoters recognized by RNA polymerase with different sigma factors were also studied. A special procedure was developed for the study of correlations between the intensities of periodicities in promoter sequences and the expression levels of corresponding genes. Significant correlations of expression with the AT content and with AT periodicities about 10, 11, and 50nt indicate their role in regulation of supercoiling-sensitive genes.

Coordination of genomic structure and transcription by the main bacterial nucleoid‐associated protein HU

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci-spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.

Homeostatic regulation of supercoiling sensitivity coordinates transcription of the bacterial genome

Regulation of cellular growth implies spatiotemporally coordinated programmes of gene transcription. A central question, therefore, is how global transcription is coordinated in the genome. The growth of the unicellular organism Escherichia coli is associated with changes in both the global superhelicity modulated by cellular topoisomerase activity and the relative proportions of the abundant DNA-architectural chromatin proteins. Using a DNA-microarray-based approach that combines mutations in the genes of two important chromatin proteins with induced changes of DNA superhelicity, we demonstrate that genomic transcription is tightly associated with the spatial distribution of supercoiling sensitivity, which in turn depends on chromatin proteins. We further demonstrate that essential metabolic pathways involved in the maintenance of growth respond distinctly to changes of superhelicity. We infer that a homeostatic mechanism organizing the supercoiling sensitivity is coordinating the growth-phase-dependent transcription of the genome.

Genome-wide prediction of G4 DNA as regulatory motifs: Role inEscherichia coliglobal regulation

The role of nonlinear DNA in replication, recombination, and transcription has become evident in recent years. Although several studies have predicted and characterized regulatory elements at the sequence level, very few have investigated DNA structure as regulatory motifs. Here, using G-quadruplex or G4 DNA motifs as a model, we have researched the role of DNA structure in transcription on a genome-wide scale. Analyses of >61,000 open reading frames (ORFs) across 18 prokaryotes show enrichment of G4 motifs in regulatory regions and indicate its predominance within promoters of genes pertaining to transcription, secondary metabolite biosynthesis, and signal transduction. Based on this, we predict that G4 DNA may present regulatory signals. This is supported by conserved G4 motifs in promoters of orthologous genes across phylogenetically distant organisms. We hypothesized a regulatory role of G4 DNA during supercoiling stress, when duplex destabilization may result in G4 formation. This is in line with our observations from target site analysis for 55 DNA-binding proteins in Escherichia coli, which reveals significant (P<0.001) association of G4 motifs with target sites of global regulators FIS and Lrp and the sigma factor RpoD (sigma70). These factors together control >1000 genes in the early growth phase and are believed to be induced by supercoiled DNA. We also predict G4 motif-induced supercoiling sensitivity for >30 operons in E. coli, and our findings implicate G4 DNA in DNA-topology-mediated global gene regulation in E. coli.

Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity.

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3' to the TATA box within an 11 bp alternating purine-pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5' to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5' to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3' end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.

The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control.

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich. Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

Regulation of the Escherichia coli nrd operon: role of DNA supercoiling

An in vitro RNA transcription assay was used to investigate the regulation of the expression of the nrd promoter. Using a linear DNA template, we found that Fis protein, which has a positive effect on expression of the nrd promoter in an nrd-lacZ fusion in vivo, had a moderate negative effect in vitro. However, with a supercoiled DNA template as substrate, we found that Fis had a concentration-dependent positive effect on nrd transcription in vitro. This positive effect was not present on two templates that had 35- or 37-bp insertions between the Fis binding site and the nrd promoter. In the absence of Fis protein, a dramatic decrease in transcription was observed in templates with reduced supercoiling generated by the treatment with wheat germ topoisomerase I. Templates with insertions of 35 bp into an HpaII site at -102 or 37 bp into the MnlI site at -33 bp from the start of transcription failed to exhibit the DNA supercoiling sensitivity of the nrd promoter. Analysis of cells containing either of these two nrd-lacZ fusion constructs that has an insertion at the regulatory region by flow cytometry indicated that these two constructs, unlike the parental construct, were not cell cycle regulated.

The function of the upstream region of the σ;54‐dependent Klebsiella pneumoniae nifL promoter is sensitive to DNA supercoiling

The positive control protein NTRC activates transcription from the sigma 54-dependent nifL and glnAp2 promoters of Klebsiella pneumoniae by binding to upstream enhancer-like sequences and contacting downstream bound sigma 54-RNA polymerase via looping of the intervening DNA. In contrast to the glnAp2 promoter, the activity of the nifL promoter is very sensitive to changes in DNA supercoiling both in vivo and in vitro. We have shown previously that the downstream elements of the nifL promoter are involved in the supercoiling response. In this study we find that the upstream region of nifL influences the supercoiling response of a hybrid nifL-glnAp2 promoter both in vivo and in vitro, demonstrating that the nifL upstream region also confers supercoiling sensitivity. DNA supercoiling did not appear to influence binding of NTRC to its sites in the nifL upstream region, suggesting that another function of this region, most probably DNA loop formation, is sensitive to changes in DNA topology.

Supercoiling response of a bacterial tRNA gene.

The effect of DNA supercoiling on transcription of a bacterial tRNA gene has been analysed in vitro and in vivo. The Escherichia coli tyrT gene is found to be completely dependent on negative supercoiling when transcription is assayed in vitro at physiological salt concentrations and this supercoiling dependence is shown to be a property of the primary promoter sequences. The supercoiling sensitivity can however be removed by reducing the salt concentration below 50 mM KCl. The effect of supercoiling in vivo was analysed by measuring the activity of the tyrT promoter on a galK fusion vector in E. coli strains that have mutations in either the DNA gyrase or topoisomerase I genes. Surprisingly, in view of the dramatic in vitro effects, supercoiling could be either increased or decreased relative to the wild-type in vivo level without causing any change in tyrT promoter activity.