Structural dynamics of the active center of multisubunit RNA polymerases during RNA synthesis and proofreading

Pupov, Danil; Kulbachinskiy, Andrey

doi:10.1134/s0026893310040023

Mol Biol

2010

DOI: 10.1134/s0026893310040023

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Structural dynamics of the active center of multisubunit RNA polymerases during RNA synthesis and proofreading

Danil Pupov

Andrey Kulbachinskiy

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Cited by 2 publications

(1 citation statement)

References 107 publications

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“…Experimental data were analyzed by usage of the standard program packets "Origin 7.5" (OriginLab Corp.) and "Statistica 6" (StatSoft Inc. or ribonuclease, also depend upon Mg 2+ . Moreover, there are the specialized proteins that regulate homeo stasis and transport of Mg 2+ in cells [9,10]. However, in the available literature, up to date there have no been findings of MIE in any enzyme processes except the ATP synthesis in the above cited papers [5,6].…”

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confidence: 96%

Magnetic-isotope effect of magnesium in the living cell

Koltover

Shevchenko

Авдеева

et al. 2012

Dokl Biochem Biophys

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12 1 There is a great variety of chemical elements which have both kinds of stable isotopes, nonmagnetic and magnetic ones. For example, among three stable iso topes of magnesium, 24 Mg, 25 Mg, and 26 Mg with natu ral abundance of approximately 78.99, 10.00, and 11.01%, 25 Mg is magnetic isotope (it has nuclear spin I = 5/2) while 24 Mg and 26 Mg are nonmagnetic iso topes (nuclear spin I = 0) [1]. In this work we studied the influence of different isotopes of magnesium on cells of Escherichia coli and found that the cells which were grown on magnetic isotope, 25 Mg, essentially faster adapt to the new media by comparison with the cells which were grown on the nonmagnetic isotopes. Thus, we have experimentally revealed, for the first time, the magnetic isotope effect in vivo.The solutions of the magnesium sulphate were pre pared from the relevant oxides, 24 MgO, 25 MgO, and 26 MgO, with the isotope enrichment, correspondingly, 99.8, 98.8, and 97.7 atomic percent (Federal State Unitary Enterprise "Integrated Plant Electrohimpri bor," RosAtom). Experiments were performed with bacteria E. coli, "museum" strain K12TG1, which were cultivated on the solid lactose BCP agar (Bio Merieux, France) in accordance with the standard design [2, 3]. The bacterial inoculates have been pre viously incubated for 24 h in the artificial liquid nutri ent medium M9, that was composed from 8 g of glu cose, 2 g of NH 4 Cl, 12 g of Na 2 HPO 4 , 6 g of K 2 HPO 4 , 1 g of NaCl in 1 liter of distilled water, pH 7.0, without magnesium. Thereafter, the equal aliquots of the cells (400 µL) were suspended in 50 mL of the same medium but supplemented with 24 Mg, 25 Mg, or 26 Mg 1 The article was translated by the authors. as 2.2 mM of 24 MgSO 4 , 25 MgSO 4 , or 26 MgSO 4 , and were then cultivated under the conditions of continu ous aeration with shaking at 37°C. The experiments with different isotopes were performed simulta neously, three bacteria samples with each kind of the isotope, a total of 9 samples under the same conditions in parallel. According to the data of high resolution mass spectrometry (spectrometer ICP-MS "Ele ment-2," USA), the element compositions of the media were the same for all samples with amounts of contaminants no more than 10 -6 M, no matter which kind of magnesium isotope was used. Kinetics of the cell growth was hourly monitored by optical density measurements at 620 nm (OD 620 ) using multi channel immunoenzyme analyzer "Uniplan" (Picon, Russia). The colony forming ability of bacteria was tested by the routine technique of seeding the cells, which entered the stationary phase of growth, on the solid nutrient media (BCP-agar) accompanied by count ing the colonies after incubation for 16 h at 37°C [2, 3]. The experimental sessions were replicated twice with different microbial inoculates.The kinetic curves of growth of the cell cultures in our experiments were of the standard form: the phase of relatively slow adaptation to the new growth media ("lag-phase") followed by the rapid phase of expo nential growth ("log-ph...

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mentioning

confidence: 96%

Magnetic-isotope effect of magnesium in the living cell

Koltover

Shevchenko

Авдеева

et al. 2012

Dokl Biochem Biophys

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Purification and characterization of recombinant Deinococcus radiodurans RNA Polymerase

Esyunina

Kulbachinskiy

2015

Biochemistry Moscow

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The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

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Structural dynamics of the active center of multisubunit RNA polymerases during RNA synthesis and proofreading

Cited by 2 publications

References 107 publications

Magnetic-isotope effect of magnesium in the living cell

Magnetic-isotope effect of magnesium in the living cell

Purification and characterization of recombinant Deinococcus radiodurans RNA Polymerase

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