Direct evidence for the effect of transcription on local DNA supercoiling in vivo. Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies. Transcription generates positively and negatively supercoiled domains in the template.
Zhi X, Leng F. Dependence of transcription-coupled DNA supercoiling on promoter strength in Escherichia coli topoisomerase I deficient strains. A sign inversion mechanism for enzymatic supercoiling of DNA. Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity.
Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque. Nat Struct Mol Biol. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Williams NL, Maxwell A. Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking. Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli.
J Biol Chem. Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid.
Rates of gyrase supercoiling and transcription elongation control supercoil density in a bacterial chromosome. PLoS Genet. Single-molecule imaging of DNA gyrase activity in living Escherichia coli.
Genes Dev. Replication-transcription conflicts generate R-loops that orchestrate bacterial stress survival and pathogenesis. Kuzminov A. When DNA topology turns deadly - RNA polymerases dig in their R-loops to stand their ground: new positive and negative super twists in the replication-transcription conflict. Trends Genet ;34 2 — Cell ; 4 — Lang KS, Merrikh H. The clash of macromolecular titans: replication-transcription conflicts in bacteria.
Annu Rev Microbiol. Hypernegative supercoiling of the DNA template during transcription elongation in vitro. Coupling DNA supercoiling to transcription in defined protein systems. A microarray-based antibiotic screen identifies a regulatory role for supercoiling in the osmotic stress response of Escherichia coli.
Genome Res. Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli. Genome Biol. Effect of varying the supercoiling of DNA on transcription and its regulation. Control of virulence gene transcription by indirect readout in Vibrio cholerae and Salmonella enterica serovar typhimurium.
Environ Microbiol. Transcription elongation. Regulation of transcription elongation and termination. Transcription is regulated by NusA:NusG interaction. Bacterial transcription elongation factors: new insights into molecular mechanism of action. Maintenance of transcription-translation coupling by elongation factor P mBio ;7:e—e Sci Rep.
Menzel R, Gellert M. Regulation of the genes for E. Unniraman S, Nagaraja V. Regulation of DNA gyrase operon in Mycobacterium smegmatis : a distinct mechanism of relaxation stimulated transcription. Genes Cells. The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA. Autoregulation of topoisomerase I expression by supercoiling sensitive transcription.
Tse-Dinh Y, Beran R. Multiple promoters for transcription of the E. Homeostatic regulation of supercoiling sensitivity coordinates transcription of the bacterial genome. EMBO Rep. An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene. Franco RJ, Drlica K. Gyrase inhibitors can increase gyrA expression and DNA supercoiling.
Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving gene-expression and metabolic regulation of both topoisomerase I and DNA gyrase.
Eur J Biochem. Rani P, Nagaraja V. Genome-wide mapping of topoisomerase I activity sites reveal its role in chromosome segregation. Role of DNA supercoiling and rpoS sigma factor in the osmotic and growth phase-dependent induction of the gene osmE of Escherichia coli K The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity.
Hengge R. Res Microbiol. Battesti A, Bouveret E. Bougdour A, Gottesman S. Diversity in p ppGpp metabolism and effectors.
Curr Opin Microbiol. Potrykus K, Cashel M. Iron limitation induces SpoT-dependent accumulation of ppGpp in Escherichia coli. Genetically separable functional elements mediate the optimal expression and stringent regulation of a bacterial tRNA gene.
Mizushima-Sugano J, Kaziro Y. Regulation of the expression of the tufB operon: DNA sequences directly involved in the stringent control.
Travers AA. Promoter sequence for stringent control of bacterial ribonucleic acid synthesis. Alteration of the growth-rate-dependent regulation of Escherichia coli tyrT expression by promoter mutations. A proximal promoter element required for positive transcriptional control by guanosine tetraphosphate and DksA protein during the stringent response. Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli.
Changes associated with a transition to anaerobic growth. J Mol Biol ; 3 — Energy buffering of DNA structure fails when Escherichia coli runs out of substrate. Cell Biophys. Isolation and characterization of adenylate kinase adk mutations in Salmonella typhimurium which block the ability of glycine betaine to function as an osmoprotectant.
A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. Autoinduction of the ompR response regulator by acid shock and control of the Salmonella enterica acid tolerance response. Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar typhimurium during adaptation to acid stress. Karem K, Foster JW. The influence of DNA topology on the environmental regulation of a pH-regulated locus in Salmonella typhimurium.
Bacterial regulon evolution: distinct responses and roles for the identical OmpR proteins of Salmonella typhimurium and Escherichia coli in the acid stress response.
Roles for DNA supercoiling and the Fis protein in modulating expression of virulence genes during intracellular growth of Salmonella enterica serovar typhimurium. Mol Microbiol ;62 3 — DNA supercoiling and osmoresistance in Bacillus subtilis Curr Microbiol. Meury J, Kohiyama M. Potassium ions and changes in bacterial DNA supercoiling under osmotic stress. The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity.
Osmotic and growth-phase dependent regulation of the eta gene of Staphylococcus aureus : a role for DNA supercoiling. Mol Gen Genet. Escherichia coli response to hydrogen peroxide: a role for DNA supercoiling, topoisomerase I and Fis. A role for DNA supercoiling in the regulation of the cytochrome bd oxidase of Escherichia coli.
Transmission of an oxygen availability signal at the Salmonella enterica serovar typhimurium fis promoter. PLoS One. Cortassa S, Aon MA. Altered topoisomerase activities may be involved in the regulation of DNA supercoiling in aerobic-anaerobic transitions in Escherichia coli.
Mol Cell Biochem. During alkaline lysis plasmid preps, plasmids are denatured because the hydrogen bonds are disrupted by the alkaline conditions.
But the covalently closed circular strands remain intact and topologically constrained and when the pH is returned to neutral the hydrogen bonds reform and the supercoiled DNA is re-formed. However, if the alkaline lysis step is overly harsh e. Although DNA plasmid preps can return multiple forms of DNA, there is only one kind you want for successful cloning and transfection: supercoiled.
Make sure you know how to increase your recovery of good quality supercoiled DNA. Did this help you understand why you get three bands when running plasmid DNA on agarose gels? Do you have any other plasmid prep tips? Has this helped you? Then please share with your network. Does oc dna with smaller size than ccc dna run faster? Facebook Twitter LinkedIn More. Written by Dr Rebecca Tirabassi. You can see this very clearly in lane 7, where restriction fragments originating from one microgram of identical DNA molecules are separated.
Which means that the bands contain equimolar amounts DNA. The smallest fragment of basepairs 1 is hardly visible, while the biggest fragment of more than Band 3 contains smaller DNA fragments than band 2, but is still much brighter. This is because there is more nanograms of DNA in 3 than in 2 the number of molecules in 3 must be much higher than in 2.
Plasmid DNA can exist in three conformations: supercoiled, open-circular oc , and linear supercoiled plasmid DNA is often referred to as covalently closed circular DNA, ccc. In vivo, plasmid DNA is a tightly supercoiled circle to enable it to fit inside the cell.
In the laboratory, following a careful plasmid prep, most of the DNA will remain supercoiled, but a certain amount will sustain single-strand nicks.
Given the presence of a break in only one of the strands, the DNA will remain circular, but the break will permit rotation around the phosphodiester backbone and the supercoils will be released. A small, compact supercoiled knot of ccc-DNA sustains less friction against the agarose matrix than does a large, floppy open circle of oc-DNA.
Linear DNA runs through a gel end first and thus sustains less friction than open-circular DNA, but more than supercoiled. Thus, an uncut plasmid produces two bands on a gel, representing the oc and ccc conformations.
If the plasmid is cut once with a restriction enzyme, however, the supercoiled and open-circular conformations are all reduced to a linear conformation. Following isolation, spontaneous nicks accumulate as a plasmid prep ages. This can clearly be seen on gels as the proportion of the two conformations change over time: plasmids preps that have been thawed and refrozen many times, show more oc DNA than fresh preps.
This is a black-and-white photograph of an agarose gel containing ethidium bromide, after electrophoresis of three DNA samples.
0コメント