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Mg 2+ Does not induce isomerization of the open transcription complex Escherichia coli RNA polymerase at the model Pα promoter bearing consensus -10 and -35 hexamers.

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Kolasa I. K., Łoziński T., Wierzchowski K. L.

Acta Biochimica Polonica

2001

vol. 48

issue 4

985-994

The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pα promoter bearing consensus -10 and -35 recognition hexamers were studied in vitro. Previously, this promoter was used as a control one in studies on the effect of DNA bending by An·Tn sequences on transcription initiation and shown to be fully functional in E. coli (Łoziński et al., 1991, Nucleic Acids Res. 19, 2947; Łoziński & Wierzchowski, 1996, Acta Biochim. Polon. 43, 265). The data now obtained demonstrate that the mechanism of Pα-RPo formation and dissociation conforms to the three-step reaction model: bind-nucleate-melt, commonly accepted for natural promoters. Measurements of the dissociation rate constant of Pα -RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg≈ 4, being bound to the RPo in the course of renaturation of the melted DNA region. This number was found constant in the temperature range of 25-37°C, which indicates that under these conditions the complex remaines fully open. This observation, taken together with the recent evidence from independent of the presence of Mg2+ ions (Łoziński & Wierzchowski, 2001, Acta KMnO4 footprinting studies that the length of the melted region in Pα-RPo at 37°C is Biochim. Polon. 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the λPR-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).

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1 Acta Biochimica Polonica

1 2001

1 Kolasa I. K.

1 Wierzchowski K. L.

1 Łoziński T.

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Mg 2+ Does not induce isomerization of the open transcription complex Escherichia coli RNA polymerase at the model Pα promoter bearing consensus -10 and -35 hexamers.