[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.

Gains and unexpected lessons from genome-scale promoter mapping.

Potential promoters in the genome of Escherichia coli were searched by pattern recognition software PlatProm and classified on the basis of positions relative to gene borders. Beside the expected promoters located in front of the coding sequences we found a considerable amount of intragenic promoter-like signals with a putative ability to drive either antisense or alternative transcription and revealed unusual genomic regions with extremely high density of predicted transcription start points (promoter 'islands'), some of which are located in coding sequences. PlatProm scores converted into probability of RNA polymerase binding demonstrated certain correlation with the enzyme retention registered by ChIP-on-chip technique; however, in 'dense' regions the value of correlation coefficient is lower than throughout the entire genome. Experimental verification confirmed the ability of RNA polymerase to interact and form multiple open complexes within promoter 'island' associated with appY, yet transcription efficiency was lower than might be expected. Analysis of expression data revealed the same tendency for other promoter 'islands', thus assuming functional relevance of non-productive RNA polymerase binding. Our data indicate that genomic DNA of E. coli is enriched by numerous unusual promoter-like sites with biological role yet to be understood.

[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.

Flexible elements in the structure of promoter DNA as probed by cationic surfactant binding.

A susceptibility of promoter DNA for adaptive conformational transitions has been studied using a cationic surfactant dodecyltrimethylammonium bromide (C(12)TAB) as a model DNA-binding ligand. DNAse 1 and KMnO(4) were utilized as structure-specific reagents. Both reagents revealed ligand-induced perturbations in the double helix of promoters T7A1 and T7D. These conformational transitions appeared to be strongly associated with pyrimidine-purine steps, which have non-random distribution within RNA polymerase contact region of the promoter DNA and are present in the binding sites for a majority of transcription regulation proteins. Potential flexibility of these elements creates therefore a specific media for transcription complex formation. Molecular mechanism of DNA interaction with C(12)TAB is discussed.

[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.

Structural organization and phase behavior of DNA-calcium-dipalmitoylphosphatidylcholine complex.

Freeze-fracture study of ultrastructure of DNA--calcium--dipalmitoylphosphatidylcholine (DPPC) complex was carried out at different temperatures. For high-speed cryofixation from controllable initial temperatures, a special thermostatic chamber was designed. The fracture surface of the complex was found to be considerably different from the initial DPPC liposomes: 1) the period of ripple phase was 25 nm in contrast to 15 nm for control samples; 2) the ripple phase was observed at temperatures ranging from 6 degreesC to lipid melting temperature; 3) at temperature above the lipid melting unordered worm-like folds were formed on the fracture surface. Their length was correlated with the length of DNA fragments used in the experiment. We suppose that DNA molecules adsorbed on the membrane surface were segregated to clusters, resulting in formation of a new phase with specific structure and properties.

Interaction of bacterial RNA-polymerase with two different promoters of phage T7 DNA. Conformational analysis.

Using a rifampicin-resistant RNA polymerase with altered specificity to different promoters, the D promoter of T7 phage DNA with increased affinity to the mutant enzyme was chosen. This promoter and the T7 A1 promoter with unchanged affinity as well as some nonpromoter DNA fragments were used to compare temperature-induced conformational transitions of RNA polymerase in the course of complex formation. Conformational alterations of RNA polymerase were monitored by the fluorescent label method. It was shown that RNA polymerase undergoes a set of conformational transitions during complex formation with each promoter, some of which were similar by the character of change to spectral parameters of the label (reflecting RPi and, probably, RPo formation). The local structure of complexes formed above 33 degrees C differs for A1 and D. The conformational analysis reveals at least one temperature-dependent stage upon nonspecific interaction of the enzyme with nonpromoter DNA at 13-16 degrees C. Models of functional organization of the enzyme recognizing center and some features of the structure of the promoters which may be essential for their recognition are discussed.