Spatial organization of RNA polymerase and its relationship with transcription in Escherichia coli.

Recent studies have shown that RNA polymerase (RNAP) is organized into distinct clusters in Escherichia coli and Bacillus subtilis cells. Spatially organized molecular components in prokaryotic systems imply compartmentalization without the use of membranes, which may offer insights into unique functions and regulations. It has been proposed that the formation of RNAP clusters is driven by active ribosomal RNA (rRNA) transcription and that RNAP clusters function as factories for highly efficient transcription. In this work, we examined these hypotheses by investigating the spatial organization and transcription activity of RNAP in E. coli cells using quantitative superresolution imaging coupled with genetic and biochemical assays. We observed that RNAP formed distinct clusters that were engaged in active rRNA synthesis under a rich medium growth condition. Surprisingly, a large fraction of RNAP clusters persisted in the absence of high rRNA transcription activities or when the housekeeping σ70 was sequestered, and was only significantly diminished when all RNA transcription was inhibited globally. In contrast, the cellular distribution of RNAP closely followed the morphology of the underlying nucleoid under all conditions tested irrespective of the corresponding transcription activity, and RNAP redistributed into dispersed, smaller clusters when the supercoiling state of the nucleoid was perturbed. These results suggest that RNAP was organized into active transcription centers under the rich medium growth condition; its spatial arrangement at the cellular level, however, was not dependent on rRNA synthesis activity and was likely organized by the underlying nucleoid.

Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R) and O(L) (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

Gene silencing by H-NS from distal DNA site.

In the modern concept of gene regulation, 'DNA looping' is the most common underlying mechanism in the interaction between RNA polymerase (RNAP) and transcription factors acting at a distance. This study demonstrates an additional mechanism by which DNA-bound proteins communicate with each other, by analysing the bacterial histone-like nucleoid-structuring protein (H-NS), a general transcriptional silencer. The LEE5 promoter (LEE5p) of enteropathogenic Escherichia coli was used as a model system to investigate the mechanism of H-NS-mediated transcription repression. We found that H-NS represses LEE5p by binding to a cluster of A tracks upstream of -114, followed by spreading to a site at the promoter through the oligomerization of H-NS molecules. At the promoter, the H-NS makes a specific contact with the carboxy terminal domain of the α subunit of RNAP, which prevents the processing of RNAP-promoter complexes into initiation-competent open promoter complexes, thereby regulating LEE5p from distance.

Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation.

The host innate immune response is the first line of defense against pathogens and is orchestrated by the concerted expression of genes induced by microbial stimuli. Deregulated expression of these genes is linked to the initiation and progression of diseases associated with exacerbated inflammation. We identified topoisomerase 1 (Top1) as a positive regulator of RNA polymerase II transcriptional activity at pathogen-induced genes. Depletion or chemical inhibition of Top1 suppresses the host response against influenza and Ebola viruses as well as bacterial products. Therapeutic pharmacological inhibition of Top1 protected mice from death in experimental models of lethal inflammation. Our results indicate that Top1 inhibition could be used as therapy against life-threatening infections characterized by an acutely exacerbated immune response.