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1

Evaluation of mixed-salt effects on thermodynamic and kinetic parameters of RNA polymerase-promoter DNA complexes in terms of equivalent salt concentrations. General applicability to DNA complexes

100%

Łoziński T., Wierzchowski K.

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issue 4

695-702

EN

Facile evaluation of mixed-salt effect on the strongly salt-dependent thermodynamic and kinetic parameters of protein-DNA complexes is of importance for relevant biochemical and biophysical studies. In pursuit of this aim, binding isotherms for open transcription complex (RPo) of Escherichia coli RNA polymerase (R) at λPR promoter DNA (P) were determined as a function of salt concentration in pure NaCl and Tris/HCl solutions, and as a function of [NaCl] in the presence of fixed concentrations of MgCl2 and Tris/HCl. A concept of equivalent salt concentrations, i.e. concentrations at which the binding equilibrium constant is the same, was introduced and applied for prediction of binding isotherms in mixed salt solutions. Full coincidence between the experimental and predicted isotherms indicated that individual contributions of salts to the global salt-effect are additive in a broad range of salt concentrations. A generalized formula for calculation of salt equivalents characteristic for any of the thermodynamic or kinetic parameters of a complex (e.g., free energy, binding equilibrium and association/dissociation kinetic rate constants) is presented and its applicability to a number of protein-DNA complexes and dsDNA melting demonstrated using authors' own and literature data.

2

Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic Pa promoter. A quantitative KMnO4 footprinting study

100%

Łoziński T., Wierzchowski K. L.

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2001

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vol. 48

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issue 2

495-510

EN

Footprinting studies of prokaryotic open transcription complexes (RPO), based on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxidant dose, have suggested that the extent of DNA melting in the transcription bubble region increases in the presence of Mg2+. In this work, quantitative KMnO4 footprinting in function of the oxidant dose of RPO, using Escherichia coli RNA polymerase (Eσ70 ) at a fully functional synthetic promoter Pa having -35 and -10 consensus hexamers, has been used to determine individual rate constants of oxidation of T residues in this region at 37°C in the absence of Mg2+ and in the presence of 10 mM MgCl2, and to evaluate therefrom the effect of Mg2+ on the extent of DNA melting. Population distributions of end-labeled DNA fragments corresponding to oxidized Ts were quantified and analyzed according to the single-hit kinetic model. Pseudo-first order reactivity rate constants, ki, thus obtained demonstrated that Mg2+ ions bound to RPO merely enhanced the reactivity of all 11 oxidizable thymines between the +3 and -11 promoter sites by a position-dependent factor: 3-4 for those located close to the transcription start point +1 in either DNA strand, and about 1.6 for those located more distantly therefrom. On the basis of these observations, we conclude that Mg2+ ions bound to RPO at Pa do not influence the length of the melted DNA region and propose that the higher reactivity of thymines results mainly from lower local repulsive electrostatic barriers to MnO4- diffusion around carboxylate binding sites in the catalytic center of RPO and promoter DNA phosphates.

3

Effect of reversed orientation and length of An·Tn DNA bending sequences in the -35 and spacer domains of a consensus-like Escherichia coli promoter on its strength in vivo and gross structure of the open complex in vitro

100%

Łoziński T., Wierzchowski K. L.

Acta Biochimica Polonica

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1996

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vol. 43

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issue 1

265-279

4

Effect of Mg2+ on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate.

100%

Łoziński T., Wierzchowski K. L.

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2001

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vol. 48

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issue 2

511-523

EN

Potassium permanganate oxidation of pyrimidine bases is often used to probe single-stranded regions in functional DNA-protein complexes. However, so far reactivity of these bases in double-stranded DNA has not been studied quantitatively. We have investigated the kinetics of oxidation of pyrimidines in supercoiled pDS3 plasmid dsDNA by quantitative KMnO4 footprinting, in connection with parallel studies on the effect of Mg2+ on kinetics of oxidation of individual thymines in the single-stranded region of the open transcription complex of Escherichia coli RNA polymerase at a cognate Pa promoter contained in this plasmid. Rate constants of oxidation for pyrimidines, kj, in selected regions of pDS3 DNA, including Pa promoter, were determined under single-hit reaction conditions in the absence and presence of 10 mM MgCl2. Their values appeared to be sequence-dependent and were: (i) the largest for Ts in 5'TA3' and 5'TC3' steps, while 2-4 times smaller for 5'-adjacent ones in TT(A,G,C) and TTT(A) runs, (ii) for Cs in 5'TC3' steps 2-4 fold smaller than for adjacent Ts, and (iii) in the presence of Mg2+ generally larger by a sequence-dependent factor: in 5'TC3' steps of about 2 and 4 for Ts and Cs, respectively, in 5'TA3' steps of TTA and TTTA sequences for 3'-terminal Ts of about 3, while for their 5'-neighbors of a distinctly smaller value of about 2. Comparison of kj data for corresponding Ts located between +1 and -10 regions of Pa promoter in dsDNA and in ssDNA form in the open transcription complex, reported elsewhere, demonstrates that reactivity of pyrimidines in dsDNA is by 2-3 orders of magnitude smaller. The effect of Mg2+ in dsDNA is interpreted in terms of electrostatic barrier to diffusion of MnO4- on DNA surface, which is lowered by diffusive binding of these ions to backbone phosphates, involving also sequence-specific contacts with bases in the minor and major grooves of B-DNA.

5

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

81%

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

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2003

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vol. 50

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issue 4

909-920

EN

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase α subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of σ-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

6

Effect of An tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

81%

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

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2002

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vol. 49

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issue 3

659-669

EN

In the open transcription complex (RPo), Escherichia coli RNA polymerase s70 and α subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of An DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i) A5 within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii) A5 (Pg) or A8 (Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant ka of RPo formation is: (i) by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K1, of the initial closed complex; and (ii) several-fold smaller at Pg and Pm owing to a strongly decreased value of K1. For Pm, the latter parameter was found to be dependent exponentially on four Mg2+ ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A8·T8 fragment of B'-DNA forms a smaller number of ionic contacts with the α subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase α and s70 subunits with the proximal UP subsite and the -35 region of promoters.

7

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.

81%

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

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2001

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vol. 48

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issue 4

985-994

EN

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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Evaluation of mixed-salt effects on thermodynamic and kinetic parameters of RNA polymerase-promoter DNA complexes in terms of equivalent salt concentrations. General applicability to DNA complexes

Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic Pa promoter. A quantitative KMnO4 footprinting study

Effect of reversed orientation and length of An·Tn DNA bending sequences in the -35 and spacer domains of a consensus-like Escherichia coli promoter on its strength in vivo and gross structure of the open complex in vitro

Effect of Mg2+ on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

Effect of An tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

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.