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Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity.

ABSTRACT: DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function. Removal of the CTD tail enables GyrA to introduce writhe into DNA in the absence of GyrB, an activity exhibited by other GyrA orthologs, but not by wild-type E. coli GyrA. Strikingly, a "tail-less" gyrase holoenzyme is markedly impaired for DNA supercoiling capacity, but displays normal ATPase function. Our findings reveal that the E. coli GyrA tail regulates DNA wrapping by the CTD to increase the coupling efficiency between ATP turnover and supercoiling, demonstrating that CTD functions can be fine-tuned to control gyrase activity in a highly sophisticated manner.

SUBMITTER: Tretter EM

PROVIDER: S-EPMC3365713 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity.

Tretter Elsa M EM Berger James M JM

The Journal of biological chemistry 20120328 22

DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered ...[more]

PMID: 22457353

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Project description: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 (σ(D)) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σ(C)) 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 = σ(C)+σ(D). 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 σ(D) 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.

Dataset Information

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity.

Publications

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity.

Similar Datasets

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