Open Transcription Complex Research Articles

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.

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 lambdaP(R) 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 MgCl(2) 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.

Equivalence of Mg 2+ and Na + ions in salt dependence of the equilibrium binding and dissociation rate constants of Escherichia coli RNA polymerase open complex

Conflicting experimental data on the influence of Mg 2+ ions on the salt dependence of formation/dissociation of open transcription complex (RPo) of Escherichia coli RNA polymerase led us to carry systematic measurements of the dissociation rate constant ( k d) and thermodynamic stability of complexes at λP R and Pa promoters in a broad range of [NaCl] and [MgCl 2] at 25, 31 and 37 °C, using fluorescence detected abortive transcription assay. Values of k d determined in MgCl 2 in the presence of heparin, as a commonly used anionic competitor, were shown to depend on heparin concentration whereas in NaCl this effect was not observed. Kinetics of dissociation was therefore determined in the course of salt-induced down-shift of the binding equilibrium. Salt derivatives of k d's ( n d) appeared to be similar in NaCl (~ 8.5) and MgCl 2 (~ 10) for both complexes. Isotherms of fractional occupancy of promoters by RNAP as a function of ln [salt] were shown to conform to a sigmoid Boltzman function parameterized to include binding constant of RPo and a net change ( n obs) in the number of electrolyte ions associated with complex components upon its formation/dissociation. The fitted values of n obs appeared also similar in NaCl and in MgCl 2: ~ 18 for RPo/λP R and ~ 20 for RPo/Pa, respectively. Overall unfavorable vant'Hoff enthalpy (Δ H obs) of RPo proved to be much higher in MgCl 2 than in NaCl by ca. 20 kcal/mol for both complexes, rendering them profoundly less stable in the former salt. In both salts, Δ H obs was higher by ~ 30 kcal/mol for RPo/Pa relative to RPo/λP R. Similarity of n obs and n d values for the two salts indicates thermodynamic equivalence of Mg 2+ and Na + in [salt]-controlled binding equilibrium of RPo. This finding remains in disagreement with earlier data and suggests that salt effects on open complex stability should be sought in global compensating changes in distribution of all ionic species around the interacting complex components.

H-NS Antagonism inShigella flexneriby VirB, a Virulence Gene Transcription Regulator That Is Closely Related to Plasmid Partition Factors

The VirB protein of Shigella flexneri is a positive regulator of the major virulence operons of this enteroinvasive intracellular pathogen. VirB resembles no other transcription factor but is strongly homologous to plasmid partition proteins. We found that the binding of the VirB protein to the promoter region of the icsB virulence gene induced hypersensitivity to cleavage by DNase I over a region to which the H-NS repressor protein binds and completely abolished the protection of this sequence from DNase I by H-NS. In the absence of H-NS, the VirB protein had no additive effect on the ability of the icsB promoter to form an open transcription complex, indicating that VirB is not involved in the recruitment of RNA polymerase to the promoter or in open complex formation. Similarly, VirB did not stimulate promoter function in an in vitro transcription assay but acted as an antagonist of H-NS-mediated repression. A sequence located upstream of the icsB promoter and related to cis-acting elements involved in plasmid partitioning was required for promoter derepression by VirB. Alterations to one heptameric motif within this DNA sequence attenuated VirB binding and derepression of icsB transcription.

Mg2+‐modulated KMnO4 reactivity of thymines in the open transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands

There is still a controversy over the mechanism of promoter DNA strand separation upon open transcription complex (RPo) formation by Escherichia coli RNA polymerase: is it a single or a stepwise process controlled by Mg2+ ions and temperature? To resolve this question, the kinetics of pseudo-first-order oxidation of thymine residues by KMnO4 in the -11 ... +2 DNA region of RPo at the lambdaP(R) promoter was examined under single-hit conditions as a function of temperature (13-37 degrees C) in the absence or presence of 10 mm MgCl2. The reaction was also studied with respect to thymidine and its nucleotides (TMP, TTP and TpT) as a function of temperature and [MgCl2]. The kinetic parameters, (ox)k and (ox)E(a), and Mg-induced enhancement of (ox)k proved to be of the same order of magnitude for RPo-lambdaP(R) and the nucleotides. Unlike the complex, (ox)E(a) for the nucleotides was found to be Mg-independent. The isothermal increase in (ox)k with increasing [Mg2+] was thus interpreted in terms of a simple model of screening of the negative charges on phosphate groups by Mg2+ ions, lowering the electrostatic barrier to the diffusion of MnO4- anions to the reactive double bond of thymine. Similar screening isotherms were determined for the oxidation of two groups of thymines in RPo at a consensus-like Pa promoter, differing in the magnitude of the Mg effect. Together, the findings show that: (a) the two DNA strands in the -11...+2 region of RPo-lambdaP(R) are completely separated over the whole range of temperatures investigated (13-37 degrees C) in the absence of Mg2+ (b) Mg2+ ions induce an increase in the rate of the oxidation reaction by screening negatively charged phosphate and carboxylate groups; and (c) the observed thymine reactivity and the magnitude of the Mg effect reflect variation in the strength of the electrostatic potential along the separated DNA strands, in agreement with the current structural model of RPo.

Inactivation and destruction by KMnO 4 of Escherichia coli RNA polymerase open transcription complex: recommendations for footprinting experiments

Potassium permanganate oxidation of pyrimidine residues in single-stranded DNA is commonly used in footprinting studies on formation of open transcription complex (RPo) by RNA polymerases (RNAP) at cognate promoters. Our own experience and literature search led us to conclude that KMnO 4 doses often used in such studies might cause multiple-hit oxidation of promoter DNA and oxidative damage to RNAP in RPo and lead to false interpretation of footprints. We have therefore studied as a function of KMnO 4 dose (i) transcription activity of RPo formed by Escherichia coli RNAP at a model cognate promoter Pa and (ii) RPo’s structural integrity, by gel electrophoresis and footprinting assays. Kinetics of formation of this complex and melting of DNA in the transcription bubble region were thoroughly characterized by us previously. Here we show that (i) RPo becomes completely inactivated at oxidant doses much lower than those needed to cause a detectable footprint of the melted DNA region, (ii) footprinting patterns of the melted promoter region remain practically unaffected by RNAP oxidation within a range of low oxidant doses causing single-hit oxidation of DNA, and (iii) at higher oxidant doses, corresponding to multiple-hit DNA oxidation, the gross structure of RPo changes progressively until its complete collapse and dissociation into constituent components, so that only approximate interpretation of the footprinting data for the melted DNA region is possible. A protocol for accurate RPo footprinting with low single-hit KMnO 4 doses and interpretation of the footprinting data in terms of kinetics of oxidation of pyrimidine residues in promoter DNA is recommended.

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

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha 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 A(n) 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). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(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 k(a) 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, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) 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 A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

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

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha 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 A(n) 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). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(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 k(a) 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, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) 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 A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Mg2+ does not induce isomerization of the open transcription complex of Escherichia coli RNA polymerase at the model Pa promoter bearing consensus -10 and -35 hexamers.

The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pa 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 x Tn sequences on transcription initiation and shown to be fully functional in E. coli (Loziński et al., 1991, Nucleic Acids Res. 19, 2947; Loziński & Wierzchowski, 1996, Acta Biochim. Polon. 43, 265). The data now obtained demonstrate that the mechanism of Pa-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 Pa-RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg approximately/= 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 degrees C, which indicates that under these conditions the complex remaines fully open. This observation, taken together with the recent evidence from KMnO4 footprinting studies that the length of the melted region in Pa-RPo at 37 degrees C is independent of the presence of Mg2+ ions (Lozinski & Wierzchowski, 2001, Acta Biochim. Polon. 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the lambdaP(R)-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).

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

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 o tinctly 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.

Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases.

Primer extension assays using recombinant templates constructed to contain all 256 possible base quartets in a minimum length sequence were used to examine binding of the anticancer drug actinomycin D to single-stranded DNA. Single-stranded templates were generated by digestion of linearized plasmid with the double-strand-specific T7 gene 6 exonuclease. Actinomycin D formed high-affinity, kinetically stable complexes that paused primer elongation at specific sites by HIV-1 reverse transcriptase, Sequenase (modified T4 DNA polymerase), the Klenow fragment of Escherichia coli DNA polymerase, and Vent (exo-) DNA polymerase. Pauses occurred most commonly near G+C-rich nucleotide clusters, including GpC steps, the preferred sites of double-stranded DNA binding. Complexes were stable for several minutes at temperatures over 50 degrees C as determined by their abilities to pause Vent polymerase at elevated temperatures. Significant variations were noted in pause patterns of different polymerases, demonstrating differential responses of polymerases to a bound actinomycin. Covalent adducts formed on template DNA by a photoaffinity analog of actinomycin D completely stopped primer extension. These results support the possibility that actinomycin D inhibits transcription elongation by complexing single-stranded DNA in the open transcription complex. Single-stranded DNA binding by actinomycin D or analogs may also provide routes for combating HIV or other viruses which replicate through single-stranded intermediates.

A common ATP requirement for open complex formation and transcription at promoters containing initiator or TATA elements.

The involvement of ATP during transcription initiation from three basic types of RNA polymerase II promoters was investigated. Hydrolysis of the beta-gamma bond of ATP was found to be required for transcription at TATA, initiator, and initiator + TATA promoters. For all three promoters, ATP is required to melt the transcription start site region during formation of a functional open transcription complex. The use of ATP to open the DNA strands occurs rapidly. The resulting open complex is transient, since the start site re-closes rapidly upon initiation. The requirements for efficient open complex formation were similar to those for transcription in that both upstream elements and a basal element, either TATA or initiator, were necessary. The results further suggest that TATA-dependent and initiator-dependent preinitiation complex assembly pathways converge, prior to a step in which the strands are rapidly opened in the presence of ATP, to form analogous open complexes.

Bending of DNA by transcription factors

An increasing number of transcription factors both from prokaryotic and eukaryotic sources are found to bend the DNA upon binding to their recognition site. Bending can easily be detected by the anomalous electrophoretic behaviour of the DNA-protein complex or by increased cyclization of DNA fragments containing the protein-induced bend. Induction of DNA bending by transcription factors could regulate transcription in various ways. Bending may bring distantly bound transcription factors closer together by facilitating DNA-looping or it could mediate the interaction between transcription factors and the general transcription machinery by formation of large nucleoprotein structures in which the DNA is wrapped around the protein complex. Alternatively, the energy stored in a protein-induced bend could be used to favour formation of an open transcription complex or to dissociate the RNA polymerase in the transition from initiation to elongation. Modification of the bend angles and bending centers, caused by homodimerization or heterodimerization of transcription factors, may well turn out to be an important way to enlarge the range of interactions required for regulation of gene expression.