DNA supercoiling in Escherichia coli is under tight and subtle homeostatic control, involving geneâexpression and metabolic regulation of both topoisomeraseâI and DNA gyrase
2002
Snoep, Jacky L. | van der Weijden, Coen C. | Andersen, Heidi W. | Westerhoff, Hans V. | Jensen, Peter Ruhdal
DNA of prokaryotes is in a nonequilibrium structural state, characterized as ‘active’ DNA supercoiling. Alterations in this state affect many life processes and a homeostatic control of DNA supercoiling has been suggested [Menzel, R. & Gellert, M. (1983) Cell34, 105–113]. We here report on a new method for quantifying homeostatic control of the highâenergy state of in vivo DNA. The method involves making small perturbation in the expression of topoisomeraseâI, and measuring the effect on DNA supercoiling of a reporter plasmid and on the expression of DNA gyrase. In a separate set of experiments the expression of DNA gyrase was manipulated and the control on DNA supercoiling and topoisomeraseâI expression was measured [part of these latter experiments has been published in Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff, H.V. & Snoep, J.L. (1999) Eur. J. Biochem.266, 865–877]. Of the two regulatory mechanisms via which homeostasis isâ£conferred, regulation of enzyme activity or regulation of enzyme expression, we quantified the first to be responsible for 72% and the latter for 28%. The gene expression regulation could be dissected to DNA gyrase (21%) and to topoisomeraseâI (7%). On a scale from 0 (no homeostatic control) to 1 (full homeostatic control) we quantified the homeostatic control of DNA supercoiling at 0.87. A 10% manipulation of either topoisomeraseâI or DNA gyrase activity results in a 1.3% change of DNA supercoiling only. We conclude that the homeostatic regulation of the nonequilibrium DNA structure in wildâtype Escherichia coli is almost complete and subtle (i.e. involving at least three regulatory mechanisms).
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