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1

The application of FPLC chromatography [Mono Q] for the purification of wheat RNA polymerases II and III

100%

Glanc P., Galbas M., Dullin P., Szalata M., Slomski R.

Cellular and Molecular Biology Letters

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2003

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tom 08

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nr 3

Transcription is the main step in the regulation of gene expression. To study this process in vitro, it is necessary to obtain highly purified RNA polymerases. Here, we describe a method of RNA polymerase purification using a Mono Q FPLC column. Using Mono Q column chromatography accelerates the purification process and separates RNA polymerase II from RNA polymerase III with good yield.

2

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

88%

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

Acta Biochimica Polonica

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2002

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tom 49

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nr 3

In the open transcription complex (RPo), Escherichia coli RNA polymerase o and a70 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, Kj, of the initial closed complex; and (ii) several-fold smaller at Pg and Pm owing to a strongly decreased value of K^. For Pm, the latter parameter was found to be dependent exponentially on four Mg 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 a subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase a and o subunits with the proximal UP subsite and the -35 region of promoters.

3

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

75%

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

Acta Biochimica Polonica

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2003

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tom 50

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nr 4

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 a 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 tf-sub-unit 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.

4

Mg2plus 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

75%

Lozinski T., Wierzchowski K. L.

Acta Biochimica Polonica

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2001

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tom 48

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nr 2

Footprinting studies of prokaryotic open transcription complexes (RP O), based on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxidant dose, have suggested that the extent of DNA melt ing in the tran scrip tion bub ble re gion increases in the presence of Mg2+. In this work, quantitative KMnO4 footprinting in func tion of the ox i dant dose of RPo, us ing EscherichiacoliRNA poly mer ase (Es7 ) at a fully func tional syn thetic pro moter Pa hav ing -35 and -10 con sen sus hexamers, has been used to de ter mine in di vid ual rate con stants of ox i da tion of T res i dues in this region at 37° C in the ab sence of Mg2+ and in the pres ence of 10 mM MgCl2, and to eval u- ate there from the ef fect of Mg on the ex tent of DNA melt ing. Pop u la tion dis tri butions of end-labeled DNA frag ments cor re spond ing to ox i dized Ts were quan ti fied and an a lyzed ac cord ing to the sin gle-hit ki netic model. Pseudo-first or der re ac tiv ity rate con stants, kx, thus ob tained dem on strated that Mg2+ ions bound to RPo merely enhanced the re ac tiv ity of all 11ox i diz able thymines be tween the +3 and -11 pro moter sites by a po si tion-dependent fac tor: 3-4 for those lo cated close to the tran scrip tion start point +1 in ei ther DNA strand, and about 1.6 for those lo cated more dis tantly there from. On the ba sis of these ob ser va tions, we con clude that Mg2+ ions bound to RPo at Pa do not in flu ence the length of the melted DNA re gion and pro pose that the higher re ac tiv ity of thymines re sults mainly from lower lo cal re pul sive elec tro static bar ri ers to MnO4- dif fu sion around carboxylate bind ing sites in the cat a lytic cen ter of RPo and pro moter DNA phos phates.

5

The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein

75%

Gabig M., Obuchowski M., Ciesielska A., Latala B., Wegrzyn A., Thomas M. S., Wegrzyn G.

Acta Biochimica Polonica

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1998

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tom 45

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nr 1

Bacteriophage λ is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase α subunit (αCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for αCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the CII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the α subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between αCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and paQ-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

6

Expression of genes 51, 27, 28 coding for proteins of the central part of bacteriophage T4 baseplate in the bacteriophage T7 promoter-RNA polymerase expression system

75%

Nieradko J., Podgorska B.

Acta Biochimica Polonica

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1993

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tom 40

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nr 2

A fragment of T4 DNA (XbaI-HindIII) comprising the genes 51,27,28, which encodes the central plug proteins was cloned into plasmid pT7-5 and p7-6 (T7 RNA polymerase expressing system). The examined genes were only overexpressed when the orientation of cloned DNA to promoter <1>10 was as follows: promoter 10 and genes 51, 27, 28. This was achieved when the fragment (Xbal-Hindlll) was cloned into plasmid pT7-5. Gene 27 and 28 were overexpressed when the intact fragment (Xbal-Hindlll) was used. The high rate of the synthesis of proteins 27 and/or 28 had a strong inhibitory effect on the level of synthesis of the product of gene 51. For the overexpression of gene 51 in this system a deletion derivate which was devoid of gene 28 and a larger fragment of gene 27 was prepared.

7

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

63%

Lozinski T., Wierzchowski K. L.

Acta Biochimica Polonica

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2009

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tom 56

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nr 4

695-702

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.

8

Comparative analysis of the gene encoding RNA-dependent RNA polymerase in RHD virus strains originating from various continents

63%

Hukowska-Szematowicz B., Lejkowska R., Deptula W.

Advances in Agricultural Sciences

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2010

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tom 13

|

nr 1

9

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Epi-genes potentiate plant biodiversity

63%

Szopa J.

BioTechnologia. Journal of Biotechnology Computational Biology and Bionanotechnology

|

2015

|

tom 96

|

nr 1

10

The expression of UVR3 and two putative photolyases, PHR2 and At4g25290, is regulated by light

63%

Sztatelman O., Labuz J., Banas A. K.

BioTechnologia. Journal of Biotechnology Computational Biology and Bionanotechnology

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2013

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tom 94

|

nr 3

11

Number and size of nucleoli in the spermatocytes of chicken and Japanese quail

63%

Andraszek K., Gryzinska M., Knaga S., Wojcik E., Smalec E.

Folia Biologica

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2012

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tom 60

|

nr 3-4

12

Transcriptional activity of a complex of wheat RNA polymerase II and nonhistone chromosomal protein (NHCP)

63%

Dullin P., Galbas M., Chodynicka-Kordus G., Fabisz-Kijowska A., Walerych W.

Acta Biochimica Polonica

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1989

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tom 36

|

nr 3-4

13

Mg2plus 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

63%

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

Acta Biochimica Polonica

|

2001

|

tom 48

|

nr 4

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 • 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 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 ~ 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 KMnO4 footprinting studies that the length of the melted region in Pa-RPo at 37°C is independent of the presence of Mg2+ ions (Łoziński & 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 λPr-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).

14

Effect of reversed orientation and length of An.Tn DNA binding 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

63%

Lozinski T., Wierzchowski K. L.

Acta Biochimica Polonica

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1996

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tom 43

|

nr 1

In continuation of an earlier study (Łoziński ct at., 1991 Nucleic Acids Res. 19, 2947-2953) a series of consensus-like E. coli promoters with bending An Tn sequences of different length (n = 3-8) and orientation in the -35 and spacer domains was constructed, cloned into the plasmid pDS3 and their strength in vivo measured in relation to an internal transcriptional standard. Gel mobilities of free DNA restriction fragments carrying these promoters and of open transcriptional complexes with cognate RNA polymerase were determined by polyacrylamide gel electrophoresis and the gross structure of the complexes interpreted in terms of the theoretically predicted superstructure of DNA restriction fragments. The results obtained together with those reported earlier show that bending of the DNA helix axis immediately upstream of the -35 domain generally lowers the promoter strength in vivo and brings about shortening of the mean square end-to-end distance between free DNA ends in the open complex in vitro. T4Í-34...-37) and Ts(-34...-38) tracts located in the nontemplate DNA strand had the largest and comparable effect on the promoter strength, while the As T5 (-37...-41) sequence in either orientation (As tract in the template or nontemplate strand) exerted a much smaller effect. Promoters with the spacer bent by about 40° but in different directions, by two An (n = 5 or 6) tracts aligned in phase with the B-DNA repeat and located either in the template or nontemplate strands, had somewhat lower strength in vivo but the gross geometry of the respective open complexes was the same as that of a control promoter with straight spacer. Implications of these findings are discussed in connection with the existing model of E. coli transcriptional open complex.

15

Effect of disulfide and sulphydryl reagents on abortive and productive elongation catalyzed by Escherichia coli RNA polymerase

63%

Radlowski M., Job D.

Acta Biochimica Polonica

|

1994

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tom 41

|

nr 4

The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using Escherichia coli RNA polymerase holoenzyme and poly[d(A-T)] as template. In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU. Incubation of RNA polymerase with 1 mM 2-mercaptoethanoi resulted in a 5-fold increase of the rate of UpApU synthesis. In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation. Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes.

16

Effect of DNA-interacting drugs on phage T7 RNA polymerase

63%

Piestrzeniewicz M., Studzian K., Wilmanska D., Plucienniczak G., Gniazdowski M.

Acta Biochimica Polonica

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1998

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tom 45

|

nr 1

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

17

Importance of luminal and mucosal zinc in the mechanism of experimental gastric ulcer healing

51%

Opoka W., Adamek D., Plonka M., Reczynski W., Bas B., Drozdowicz D., Jagielski P., Sliwowski Z., Adamski P., Brzozowski T.

Journal of Physiology and Pharmacology

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2010

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tom 61

|

nr 5

18

Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements

51%

Bonarek P., Kedracka-Krok S., Kepys B., Wasylewski Z.

Acta Biochimica Polonica

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2008

|

tom 55

|

nr 3

537-547

The in vitro formation of transcription complexes with Escherichia coli RNA polymerase was monitored using fluorescence anisotropy measurements of labeled fragments of DNA. The multicomponent system consisted of holo or core RNA polymerase (RNAP) and lac or galpromoter fragments of DNA (in different configurations), in the presence or absence of CRP activator protein (wt or mutants) with its ligand, cAMP. Values of the apparent binding constants characterizing the system were obtained, as a result of all processes taking place in the system. The interaction of the promoters with core RNAP in the absence of CRP protein was characterized by apparent binding constants of 0.67 and 1.9×106M–1 for lac166 and gal178, respectively, and could be regarded as nonspecific. The presence of wt CRP enhanced the strength of the interaction of core RNAP with the promoter, and even in the case of galpromoter it made this interaction specific (apparent binding constant 2.93×107M–1). Holo RNAP bound the promoters significantly more strongly than core RNAP did (apparent binding constants 1.46 and 40.14×106M–1 for lac166 and gal178, respectively), and the presence of CRP also enhanced the strength of these interactions. The mutation in activator region 1 of CRP did not cause any significant disturbances in the holo RNAP–lacpromoter interaction, but mutation in activator region 2 of the activator protein substantially weakened the RNAP–galpromoter interaction.

Ograniczanie wyników

The application of FPLC chromatography [Mono Q] for the purification of wheat RNA polymerases II and III

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

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

Mg2plus 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

The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein

Expression of genes 51, 27, 28 coding for proteins of the central part of bacteriophage T4 baseplate in the bacteriophage T7 promoter-RNA polymerase expression system

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

Comparative analysis of the gene encoding RNA-dependent RNA polymerase in RHD virus strains originating from various continents

Epi-genes potentiate plant biodiversity

The expression of UVR3 and two putative photolyases, PHR2 and At4g25290, is regulated by light

Number and size of nucleoli in the spermatocytes of chicken and Japanese quail

Transcriptional activity of a complex of wheat RNA polymerase II and nonhistone chromosomal protein (NHCP)

Mg2plus 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

Effect of reversed orientation and length of An.Tn DNA binding 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 disulfide and sulphydryl reagents on abortive and productive elongation catalyzed by Escherichia coli RNA polymerase

Effect of DNA-interacting drugs on phage T7 RNA polymerase

Importance of luminal and mucosal zinc in the mechanism of experimental gastric ulcer healing

Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements