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.

A collection of Rhizobium etli promoters was isolated from a genomic DNA library constructed in the promoter-trap vector pBBMCS53, by their ability to drive the expression of a gusA reporter gene. Thirty-seven clones were selected, and their transcriptional start-sites were determined. The upstream sequence of these 37 start-sites, and the sequences of seven previously identified promoters were compared. On the basis of sequence conservation and mutational analysis, a consensus sequence CTTGACN16–23TATNNT was obtained. In this consensus sequence, nine on of twelve bases are identical to the canonical Escherichia coli σ70 promoter, however the R.etli promoters only contain 6.4 conserved bases on average. We show that the R.etli sigma factor SigA recognizes all R.etli promoters studied in this work, and that E.coli RpoD is incapable of recognizing them. The comparison of the predicted structure of SigA with the known structure of RpoD indicated that regions 2.4 and 4.2, responsible for promoter recognition, are different only by a single amino acid, whereas the region 1 of SigA contains 72 extra residues, suggesting that the differences contained in this region could be related to the lax promoter recognition of SigA.

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Mapping of putative promoters within the entire genome of Escherichia coli (E. coli) by means of pattern-recognition software PlatProm revealed several thousand of sites having high probability to perform promoter function. Along with the expected promoters located upstream of coding sequences, PlatProm identified more than a thousand potential promoters for antisense transcription and several hundred very similar signals within coding sequences having the same direction with the genes. Since recently developed ChIP-chip technology also testified the presence of intragenic RNA polymerase binding sites, such distribution of putative promoters is likely to be a general biological phenomenon reflecting yet undiscovered regulatory events. Here, we provide experimental evidences that two internal promoters are recognized by bacterial RNA polymerase. One of them is located within the hns coding sequence and may initiate synthesis of RNA from the antisense strand. Another one is found within the overlapping genes htgA/yaaW and may control the production of a shortened mRNA or an RNA-product complementary to mRNA of yaaW. Both RNA-products can form secondary structures with free energies of folding close to those of small regulatory RNAs (sRNAs) of the same length. Folding propensity of known sRNAs was further compared with that of antisense RNAs (aRNAs), predicted in E. coli as well as in Salmonella typhimurium (S. typhimurium). Slightly lower stability observed for aRNAs assumes that their structural compactness may be less significant for biological function.

Horizontally acquired genes are usually transcriptionally inactive, although most of them are associated with genomic loci enriched with promoter-like sequences forming "promoter islands." We hypothesized that lateral DNA transfer induces local mutagenesis, accumulating AT base pairs and creating promoter-like sequences, whose occupancy with RNA polymerase and a specific silencer H-NS suppresses the transcription of foreign genes. Error-prone mutagenesis was implemented for the "promoter island" of a foreign gene appY and the promoter region of an inherent gene dps. Derivatives with changed transcriptional activity were selected using a reporter plasmid pET28_eGFP. Only one cycle of mutagenesis with negative selection suppressed the activity of the main dps promoter to the background level due to a single substitution in its-10 element, while positive selection gave a sequence with improved-35 element, thus testifying feasibility of the approach. The same suppression for appY was achieved by three cycles, while eightfold transcription activation required nine iterations of mutagenesis. In both cases, the number of potential start points decreased resulting in an ordinary regulatory region with only one dominant promoter in the case of positive selection. Efficiency of H-NS binding remained virtually unchanged in all mutant constructs. Based on these findings we conclude that excessive promoters can adversely affect transcription by providing a platform for interference between several RNA polymerase molecules, which can act as a silencer at promoter-dense regions.

Mosaic pattern of transcription in alternating directions is a common feature of prokaryotic and eukaryotic genomes which rationality and origin remain enigmatic. In Escherichia coli approximately 25% of genes comprise pairs of topologically linked divergently transcribed units. Given that transcriptional complex formation at each promoter in the pair induces topological changes and is itself sensitive to DNA structural perturbations, study of the functional anatomy in such areas requires special approaches. Here we suggested the dual-colour promoter probe vector which may become an ideal tool for divergent transcription profiling. The vector was used to characterize the specific genomic region nearby appY with multiple bidirectional promoters predicted in silico. Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes. RNA product transcribed in antisense direction is suggested as a novel RNA. Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.

The potentially transcribed regions in the genome of E, coli were searched for on a systematic basis using the novel pattern recognition software PlatProm. PlatProm takes into consideration both the sequence-specific and structurespecific features in the genetic environment of the promoter sites and identifies transcription start points with a very high accuracy. The whole genome scanning by PlatProm along with the expected promoters upstream of the annotated genes identified several hundred of very similar signals in other intergenic regions and in many coding sequences. Most of them are expected as start points for independent RNA transcripts, providing a unique opportunity to reveal genes encoding antisense and/or alternative RNAs. The potential PlatProm as a tool revealing sRNA genes is discussed.

mtDNA D-loop is a non-coding locus actively used as an individualizing marker in molecular genetic research. Uneven distribution of SNP in D-loop suggests about irregular functional load within this region. The structural-functional role of various sites in D-loop of single individual's mtDNA and the degree of its (functional) importance from the point of phylogenetic conservatism are considered based on the analysis of nucleotide sequences. The role of duplication of various (functional) elements of the mtDNA D-loop (TAS, ETAS, CSB-elements) affecting the increase in functional reliability of the initiation and termination systems of the mtDNA replication is discussed.