[Bacterioferritin: properties, structural and functional organization of the dps gene regulatary region].

The paper considers the properties of bacterioferritin Dps, which is involved in the sequestering of iron ions, forms the ferrihydrite core inside the protein cavity, and functions as a major nucleoid protein. Experimental evidence on the effect of microwave irradiation on the dps gene expression is presented. The structural and functional organization of its regulatory region is analyzed, and the technological prospects of bacterioferritin application for designing new materials with desired properties are discussed.

[Antisense transcription within the hns locus of Escherichia coli].

Scanning the entire genome of E. coli by means of pattern-recognition software PlatProm spotted out more than a thousand of potential start points for antisense transcription. Taking into account possible role of antisense RNAs in the cell regulatory networks, our top-priority interest was focused on the promoter-like sites found within genes of transcription regulators. One of them (hns) encodes a major nucleoid protein affecting expression pattern of many genomic loci. Several potential start points for antisense transcription were found within its coding sequence. Gel-retardation assays, potassium permanganate and DNAse I foot-printings confirmed the ability of the intragenic promoter located approximately 280 bp downstream of ATG to bind RNA polymerase. Primer extension revealed the cDNA of the expected size while Northern blot hybridization assumes the presence of aRNA among cellular RNAs. Relative abundance of antisense RNA and hns-mRNA in vivo exhibited dependence on growth conditions thus assuming existence of regulatory pathways keeping cellular concentration of these two transcripts at the optimal level.

[Specificiety of DNA-protein interactions within transcription complexes of Escherichia coli]. / Osobennosti DNK-belkovykh vzaimodeistvii v transkriptsionnykh kompleksakh Escherichia coli.

Current requirement for description of each new promoter assumes identification of all DNA-protein and protein-protein contacts important for transcription complex formation. Experimental approaches allow estimating which one of seven alternative sigma-subunits is employed for RNA synthesis and verifying transcription dependence on known regulatory proteins. Promoter sequence by itself also contains this information. That is why, the type of promoter as well as potential regulatory proteins with high probability may be proposed, if the transcription start point has been determined. Transcription activity of the promoter is usually less predictive. It depends on the specific contacts formed by sigma-subunits with correspondent conservative elements and on many other non-specific factors that are hardly taken into account. Interaction with RNA polymerase alpha-subunits seems does not require any particular functional group of nucleotides thus exemplifying non-sequence-specific binding within binary polymerase-promoter complexes. The role of this interaction in the transcription complex formation is the main subject of this survey that summarizes our own experimental results and the data of other authors. Attempts have been made to compare nucleotide sequences of the promoters recognized by different sigma-factors within putative contact regions with alpha-subunits and to discuss regulatory propensity of free alpha-subunits.

[Levels in the structural organization of Escherichia coli promoter DNA]. / Urovni strukturnoi organizatsii promotornoi DNK.

A number of additional structural elements were identified by statistic analysis of nucleotide sequences in promoters recognized by Escherichia coli RNA polymerase. Together with canonical hexanucleotides, these elements characterize different levels in the structural organization of promoter DNA. Sequence motifs exhibiting the highest statistical significance, which dominate in the contact regions with RNA polymerase alpha and sigma subunits, are considered as targets for specific interaction with RNA polymerase. A typical feature of these elements is the presence of easily deformable dinucleotides (TG, CA and TA) or tracts containing only A/T base pairs. Thus, we noticed that the frequency of occurrence of TA in the promoter DNA is essentially higher than the average value for the genome. Besides the regions of specific interaction with RNA polymerase, these dinucleotides are often located in the number of other sites periodically distributed along the promoter DNA. This preferred disposition suggests that deformable elements participate in the adaptive conformational transitions of the promoter DNA favoring optimal configuration of the transcription complex. Probably, the most important feature of promoter DNA revealed by statistic analysis is the presence of A/T-tracts regularly distributed in the wide range from -160 up to +75 relative to the transcription start point. Both of these spatially distributed elements (TA dinucleotides and A/T-tracts) are linked with canonical regions and, therefore, may contribute to the conformational or dynamic features of the transcription machinery. Having high statistic significance, these elements might be considered as additional factors discriminating the promoter DNA on the background of other nucleotide sequences in the genome.

[Distribution and functional significance of A/T tracts in promotor sequences of Escherichia coli]. / A/T-treki v strukture promotorov Escherichia coli: kharakter raspredeleniia i funktsional'noe znachenie.

Distribution of the A/T tracts described in earlier publications in the region extending from nucleotide -250 to +150 relative to the transcription initiation site of gene transcribed regions adjacent to promoter was studied. Upstream of the -35 region a succession of A/T tracts was discovered distributed at a shorter distance one from another than in analogous elements of the transcribed region (1 and 1.5 helix turns, respectively). Such a positional dependence suggests different functional manifestation of A/T tracts at different transcription steps. Single initiation using the T7D promoter mutant derivatives devoid of A/T tracts in two critical positions, +41 and , yielded shortened products of the corresponding length. One might speculate that such elements adjacent to promoter region play a significant role in transcription complex functioning.

[Sequence-determined conformational changes in the coding region of the promoter DNA on transcription complex formation]. / Determinirovannye nukleotidnoi posledovatgel'nost'iu konformatsionnye izmeneniia pri-promotorno DNK v kodiruiushchei oblasti pri obranzovanii transkriptsionnogo kompleksa.

Chemical footprinting was used to study the spatial structure of bacteriophage T7 promoter D upon formation of the transcriptionally active complex with Escherichia coli RNA polymerase. Enzyme binding was shown to induce conformational changes in sites located at positions 43 and 57, several helix turns away from the transcription start. This was the first finding of a structural deformation induced by assembly of the transcription complex. The deformation was associated with specific features of the promoter nucleotide sequence, and suggested high cooperativity in the organization of the transcription complex and substantial energy perturbations caused by the enzyme.

[Specific modification of the alpha-subunit of Escherichia coli Rna polymerase by monomercuric derivative of fluorescein mercuric acetate]. / Spetsificheskaia modifikatsiia alpha-sub''edinitsy RNK-polimerazy Escherichia coli monortutnym proizvodnym fluorestseinmerkuratsetata.

The method for specific modification of Escherichia coli RNA polymerase by a monomercuric derivative of fluorescein--fluoresceinmonomercuracetate (FMMA)--a specific reagent for SH-groups of proteins is suggested. It is shown, that in conditions of equimolar FMMA/enzyme ratio the fluorescent label interacts preferantially with a single sulfhydryl group in alpha-subunit of RNA polymerase. The mercaptide bonding formation is followed by significant alterations of all spectral parameters of FMMA, but has no effect on the kinetic parameters (KB and k2) of RNA synthesis initiation nor does it lead to inhibition of the total RNA synthesis. The modification presented may be used in structural and topological investigations of RNA polymerase functioning.

[RNA-synthesizing activity of brain chromatin in rats with experimental alloxan diabetes]. / RNK-sinteziruiushchaia sposobnost' khromatina mozgovoi tkani krys s éksperimental'nym alloksanovym diabetom.

Content of noradrenaline was decreased in neocortex and brain stem of rat brain by 70% and 50%, respectively, and that of serotonin--by 50% in the neocortex after development of the diabetic syndrome. At the same time, the RNA synthesizing ability of brain chromatin from the experimental animals was 3-fold higher as compared with controls. Catecholamines (L-DOPA, dopamine, noradrenaline) activated the RNA synthesis in controls by 40-50%, whereas in the animals with alloxane diabetes the effect was distinctly decreased. In vitro inhibitory effect of serotonin on the RNA synthesis was markedly decreased in preparations of brain chromatin from diabetic rats. Inhibitory effect of actinomycine D on the RNA synthesis was neutralized completely in controls after preincubation of the brain chromatin with L-DOPA, while only partial influence was detected in diabetic rats.

[Effect of catecholamines and serotonin on RNA-synthesizing activity in isolated nuclei and chromatin of the rat brain and liver]. / Vliianie katekholaminov i serotonina na RNK-sinteziruiushchuiu sposobnost' izolirovannykh iader i khromatina mozga i pecheni krys.

It has been shown that intraperitoneal injections of L-DOPA cause an increase in the matrix activity of chromatin and stimulate the incorporation of [3H]uridine into the nuclear fraction of rat brain cells by 35%. In vitro studies have shown that preincubation of brain chromatin with L-DOPA diminishes the inhibiting effect of actinomycin D on RNA synthesis. It has been found that the rate of RNA synthesis in vitro depends on concentrations of catecholamines (L-DOPA, dopamine, norepinephrine) and serotonin.

[RNA polymerase of a rifampicin-resistant mutant of Escherichia coli has an altered selectivity to phage T7 DNA promoters]. / RNK-polimeraza rifampitsin-ustoichivogo mutanta Escherichia coli imeet izmenennuiu selektivnost' k promotoram DNK faga T7.

The formation of complexes of RNA polymerases from E. coli W12 and its rpoB409 rifampicin resistant mutant with A1 and D promoters of T7 delta D111 DNA was studied by an abortive RNA synthesis technique. The mutation was shown to affect RNA synthesis initiation at these two promotors differentially so that the efficiency of D promotor utilization is enhanced but the use of A1 promotor is unchanged. The mutation does not interfere with the affinity of the enzyme for both initiating substrates. The results show that the change in RNA polymerase beta-subunit structure has a differential effect on the enzyme interaction with different promotors. The necessity of a classificatory approach to structure-functional analysis of promotors was proposed.

[The role of the beta-subunit of RNA-polymerase in specific recognition of promoters]. / Rol' beta-sub''edinitsy RNK-polimerazy v spetsificheskom uznavanii promotorov.

The complex formation of T7 DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W12) and its rifampicin resistant mutant with highly pleiotropic effect--rpoB409 was studied. As shown earlier rpoB409 RNA polymerase differs from the normal enzyme by the selection of RNA synthesis from early promoters of DNA from T7 and T4 phages. The change in the RNA specificity synthesis due to rpoB409 mutation was shown to occur at the stage of RNA polymerase interaction with DNA before open promoter complex formation. The data obtained together with the fact of highly pleiotropic effect of the rpoB409 mutation indicate that RNA polymerase beta-subunit takes part in specific recognition of promoters.

[Role of RNA polymerase in ensuring fidelity in copying the template during transcription in E. coli]. / Rol' RNK-polimerazy v obespechenii tochnosti kopirovaniia matritsy pri transkriptsii E. coli.

The heterogeneity of cell size of E. coli WU-36-10-11-12 and its four RNA-polymerase (rif-r) mutants with pleiotropic effect -- rpoB401, rpoB402, rpoB403 and rpoB409 was investigated for the purposeful choice of E. coli mutant with an altered fidelity of transcription. The stability of the phenotype of E. coli strains was shown to depend on the structural state of RNA polymerase. In vitro RNA-polymerase of the morphologically most unstable mutant rpoB402 incorporates non-complementary GMP or CMP on the poly [d(AT).d(AT)] template more frequently than the enzyme from the wild-type strain. The data obtained suggest that the beta-subunit of RNA-polymerase determines the fidelity of transcription and the selection of complementary nucleotides.

[Role of the beta-subunits of Escherichia coli RNA-polymerase in the regulation of gene activity]. / Rol' beta-sub" edinitsy RNK-polimerazy Escherichia coli v reguliatsii genov.

The DNA-dependent RNA-polymerase from E. coli B/r and its rif-r mutant rpoB409 with pleiotropic effect has been studied. It was shown, that multiple forms of promotor sites in T4- and T7-DNA "early" regions are recognized with different efficiences by RNA-polymerases from E. coli B/r and rpoB409. The rif-r rpoB409 mutation has been reported to affect the beta-subunit. Thus, the present data indicates that the selection of promoter sites can be controlled by the beta-subunit of RNA-polymerase.

[Role of RNA-polymerase in gene activity regulation of E. coli RNA-polymerase mutants with a pleiotropic effect. I. Physiological and biochemical studies]. / Rol' RNK-polimerazy v reguliatsii aktivnosti genov u Escherichia coli RNK-polimeraznye mutanty s pleiotropnym éffektom. I. Fiziologicheskie i biokhimicheskie issledovaniia.

Four Rifr-mutants of E. coli B/r (rpo B401, rpo B402, rpo B403, rpo B409) which differ from the wild strain in one or more phenotypic properties besides rifampicin resistance were obtained. Transfer of the mutant Rifr-alleles into the parent strain gives the latter all the properties of the mutant. This indicates that the new properties are due to the pleiotropic effect of Rifr-mutations. Biochemical studies of the properties of RNA-polymerases from the mutants and the parent showed that some new properties of the mutants could not be explained by the appearance of analogous properties in the mutant RNA-polymerase itself. They seem to be caused by alteration in functional activity of the mutant enzyme, particulary, alteration of its control properties during transcription. The function of the beta-subunit in genetic transcription is discussed.