The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid.
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ABSTRACT:
SUBMITTER: Yang D
PROVIDER: S-EPMC7237313 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:How genomes are organized within cells and how the 3D architecture of a genome influences cellular functions are significant questions in biology. A bacterial genomic DNA resides inside cells in a highly condensed and functionally organized form called nucleoid (nucleus-like structure without a nuclear membrane). The Escherichia coli chromosome or nucleoid is composed of the genomic DNA, RNA, and protein. The nucleoid forms by condensation and functional arrangement of a single chromosomal DNA with the help of chromosomal architectural proteins and RNA molecules as well as DNA supercoiling. Although a high-resolution structure of a bacterial nucleoid is yet to come, five decades of research has established the following salient features of the E. coli nucleoid elaborated below: 1) The chromosomal DNA is on the average a negatively supercoiled molecule that is folded as plectonemic loops, which are confined into many independent topological domains due to supercoiling diffusion barriers; 2) The loops spatially organize into megabase size regions called macrodomains, which are defined by more frequent physical interactions among DNA sites within the same macrodomain than between different macrodomains; 3) The condensed and spatially organized DNA takes the form of a helical ellipsoid radially confined in the cell; and 4) The DNA in the chromosome appears to have a condition-dependent 3-D structure that is linked to gene expression so that the nucleoid architecture and gene transcription are tightly interdependent, influencing each other reciprocally. Current advents of high-resolution microscopy, single-molecule analysis and molecular structure determination of the components are expected to reveal the total structure and function of the bacterial nucleoid.
| S-EPMC6907758 | biostudies-literature
Project description:The intrinsic stiffness of DNA limits its ability to be bent and twisted over short lengths, but such deformations are required for gene regulation. One classic paradigm is DNA looping in the regulation of the Escherichia coli lac operon. Lac repressor protein binds simultaneously to two operator sequences flanking the lac promoter. Analysis of the length dependence of looping-dependent repression of the lac operon provides insight into DNA deformation energetics within cells. The apparent flexibility of DNA is greater in vivo than in vitro, possibly because of host proteins that bind DNA and induce sites of flexure. Here we test DNA looping in bacterial strains lacking the nucleoid proteins HU, IHF or H-NS. We confirm that deletion of HU inhibits looping and that quantitative modeling suggests residual looping in the induced operon. Deletion of IHF has little effect. Remarkably, DNA looping is strongly enhanced in the absence of H-NS, and an explanatory model is proposed. Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling. These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.
| S-EPMC1919473 | biostudies-literature
Project description:Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαβ and their interactions that span the nanoscale and mesoscale from protein-DNA complexes to the bacterial chromosome and nucleoid structure. We determined the crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA binding modes of HUαα and HUαβ elucidate fundamental features of bacterial chromosome packing that regulate gene transcription. By combining crystal structures with solution x-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near-physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based on in vivo imaging of native unlabeled nucleoid by soft x-ray tomography upon HUβ and ectopic HUα38 expression. Structural characterization of charge-altered HUαα-DNA complexes reveals an HU molecular switch that is suitable for condensing nucleoid and reprogramming noninvasive Escherichia coli into an invasive form. Collective findings suggest that shifts between networking and cooperative and noncooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating x-ray crystal structures, x-ray scattering, mutational tests, and x-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis.
| S-EPMC4966879 | biostudies-literature
Project description:The genomic DNA of bacteria occupies only a fraction of the cell called the nucleoid, although it is not bounded by any membrane and would occupy a volume hundreds of times larger than the cell in the absence of constraints. The two most important contributions to the compaction of the DNA coil are the cross-linking of the DNA by nucleoid proteins (like H-NS and StpA) and the demixing of DNA and other abundant globular macromolecules which do not bind to the DNA (like ribosomes). The present work deals with the interplay of DNA-bridging proteins and globular macromolecular crowders, with the goal of determining the extent to which they collaborate in organizing the nucleoid. In order to answer this question, a coarse-grained model was developed and its properties were investigated through Brownian dynamics simulations. These simulations reveal that the radius of gyration of the DNA coil decreases linearly with the effective volume ratio of globular crowders and the number of DNA bridges formed by nucleoid proteins in the whole range of physiological values. Moreover, simulations highlight the fact that the number of DNA bridges formed by nucleoid proteins depends crucially on their ability to self-associate (oligomerize). An explanation for this result is proposed in terms of the mean distance between DNA segments and the capacity of proteins to maintain DNA-bridging in spite of the thermal fluctuations of the DNA network. Finally, simulations indicate that non-associating proteins preserve a high mobility inside the nucleoid while contributing to its compaction, leading to a DNA/protein complex which looks like a liquid droplet. In contrast, self-associating proteins form a little deformable network which cross-links the DNA chain, with the consequence that the DNA/protein complex looks more like a gel.
| S-EPMC10011147 | biostudies-literature
Project description:Bacterial small RNAs (sRNAs) regulate protein production by binding to mRNAs and altering their translation and degradation. sRNAs are smaller than most mRNAs but larger than many proteins. Therefore it is uncertain whether sRNAs can enter the nucleoid to target nascent mRNAs. Here, we investigate the intracellular localization of sRNAs transcribed from plasmids in Escherichia coli using RNA fluorescent in-situ hybridization. We found that sRNAs (GlmZ, OxyS, RyhB and SgrS) have equal preference for the nucleoid and cytoplasm, and no preferential localization at the cell membrane. We show using the gfp mRNA (encoding green fluorescent protein) that non-sRNAs can be engineered to have different proportions of nucleoid and cytoplasmic localization by altering their length and/or translation. The same localization as sRNAs was achieved by decreasing gfp mRNA length and translation, which suggests that sRNAs and other RNAs may enter the densely packed DNA of the nucleoid if they are sufficiently small. We also found that the Hfq protein, which binds sRNAs, minimally affects sRNA localization. Important implications of our findings for engineering synthetic circuits are: (i) sRNAs can potentially bind nascent mRNAs in the nucleoid, and (ii) localization patterns and distribution volumes of sRNAs can differ from some larger RNAs.
| S-EPMC5389542 | biostudies-literature
Project description:Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli. HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro. We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known.
| S-EPMC3486375 | biostudies-literature
Project description:The Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells. Systematic analysis of the nucleoid-associated proteins (NAPs) and related proteins revealed that HU, the most abundant NAP, and SlmA, the nucleoid occlusion factor regulating the localization of cell division apparatus, promote the specific localization of CrfC foci. When the replication initiator DnaA was inactivated, SlmA and HU were required for formation of CrfC foci. In contrast, when the replication initiation was inhibited with a specific mutant of the helicase-loader DnaC, CrfC foci were sustained independently of SlmA and HU. H-NS, which forms clusters on AT-rich DNA regions, promotes formation of CrfC foci as well as transcriptional regulation of crfC. In addition, MukB, the chromosomal structure mainetanice protein, and SeqA, a hemimethylated nascent DNA region-binding protein, moderately stimulated formation of CrfC foci. However, IHF, a structural homolog of HU, MatP, the replication terminus-binding protein, Dps, a stress-response factor, and FtsZ, an SlmA-interacting factor in cell division apparatus, little or only slightly affected CrfC foci formation and localization. Taken together, these findings suggest a novel and unique mechanism that CrfC localizes to the nucleoid poles in two steps, assembly and recruitment, dependent upon HU, MukB, SeqA, and SlmA, which is stimulated directly or indirectly by H-NS and DnaA. These factors might concordantly affect specific nucleoid substructures. Also, these nucleoid dynamics might be significant in the role for CrfC in chromosome partition.
| S-EPMC6374313 | biostudies-literature
Project description:HU is a conserved prokaryotic nucleoid-associated protein known for its role in DNA binding and maintaining negative supercoils in DNA. HU also binds to a few RNAs but the natural targets are unknown. To understand the biology of HU interactions with RNA in Escherichia coli, potential HU-RNA in vivo complexes were immunoprecipitated and bound RNAs were examined by hybridization to whole-genome tiling arrays. We observed HU associations with rRNA, multiple tRNAs, a few sRNAs and a small, distinct set of mRNAs. More importantly, we also identified associations between HU and ten intragenic non-coding (nonc) RNAs, two of which are homologous to the annotated Bacterial Interspersed Mosaic Elements (BIME) and boxC elements; the other eight RNAs have not been annotated. We confirmed binding of HU to BIME RNA in vitro. Since HU and some unidentified RNAs have been implicated in the nucleoid structure, we explored an idea that HU, along with some or all of the nonc RNA species discovered here bind to BIME and/or boxC repeats to condense chromosomal DNA in a specific fashion. We propose that nonc RNAs may interact with both HU and DNA repeats to form ternary complexes, which are the anchoring points separating different chromosomal domains.
2012-12-07 | GSE28565 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2017-12-25 | GSE87118 | GEO
Project description:The Escherichia coli K-12 nir operon promoter can be fully activated by binding of the regulator of fumarate and nitrate reduction (FNR) to a site centered at position -41.5 upstream of the transcript start, and this activation is modulated by upstream binding of the integration host factor (IHF) and Fis (factor for inversion stimulation) proteins. Thus, transcription initiation is repressed by the binding of IHF and Fis to sites centered at position -88 (IHF I) and position -142 (Fis I) and activated by IHF binding to a site at position -115 (IHF II). Here, we have exploited mutational analysis and biochemistry to investigate the actions of IHF and Fis at these sites. We show that the effects of IHF and Fis are position dependent and that IHF II functions independently of IHF I and Fis I. Using in vitro assays, we report that IHF and Fis repress transcription initiation by interfering with RNA polymerase binding. Differences in the upstream IHF and Fis binding sites at the nir promoter in related enteric bacteria fix the level of nir operon expression under anaerobic growth conditions.
| S-EPMC2580697 | biostudies-literature