Rates of gyrase supercoiling and transcription elongation control supercoil density in a bacterial chromosome
| Autor: | Olga N. Chesnokova, Andrews Akwasi Agbleke, Zhenhua Pang, N. Patrick Higgins, Nikolay Rovinskiy |
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| Rok vydání: | 2012 |
| Předmět: |
DNA
Bacterial Salmonella typhimurium Cancer Research Tn3 transposon Transcription Elongation Genetic lcsh:QH426-470 Biology DNA gyrase Recombinases 03 medical and health sciences chemistry.chemical_compound Transcription (biology) Genes Reporter RNA polymerase Genetics Escherichia coli Molecular Biology Genetics (clinical) Ecology Evolution Behavior and Systematics 030304 developmental biology Nucleic Acid Synthesis Inhibitors 0303 health sciences Binding Sites 030306 microbiology DNA Superhelical Circular bacterial chromosome Physics DNA-Directed RNA Polymerases Gene Expression Regulation Bacterial Chromosomes Bacterial Molecular biology lcsh:Genetics Protein Subunits Chemistry chemistry Lac Operon Prokaryotic DNA replication DNA Gyrase Mutation Biophysics DNA supercoil Rifampin DNA Mathematics Protein Binding Research Article |
| Zdroj: | PLoS Genetics PLoS Genetics, Vol 8, Iss 8, p e1002845 (2012) |
| ISSN: | 1553-7404 |
| Popis: | Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound “nucleoid.” The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σD) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σC) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σC+σD. Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb “supercoil sensor” inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (−) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σD at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species. Author Summary A 9-kb module called the “supercoil sensor” was used to measure supercoil density at 10 positions in the 4.8-Mb Salmonella Typhimurium chromosome. The sensor includes a Lac operon flanked by a pair of directly repeated DNA–binding sites for the γδ recombinase. Measurements of chromosomal supercoil levels and the RNA polymerase elongation rates were made at various positions within the 6 potential macrodomains of the chromosome. Transcription and gyrase catalytic rates were mechanistically linked. Gyrase mutants with impaired activity caused the loss of from 30% to >95% of the diffusible supercoiling throughout most of the chromosome, while treatment with rifampicin that temporarily blocked transcription restored most of the lost supercoils in gyrase mutants. A gyrase defect also caused transcription elongation rates to decrease across the chromosome, and a mutation that reduced RNA polymerase efficiency increased average chromosome supercoiling levels. A model in which topoisomerases act close to highly transcribed operons to equilibrate the supercoil flux generated by transcription suggests that matched rates of gyrase turnover and transcription elongation speed determine the average supercoil density in bacterial chromosomes. |
| Databáze: | OpenAIRE |
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