RESUMEN
The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s).
Asunto(s)
Endopeptidasas , Escherichia coli , Operón , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Transcripción Genética , Proteínas Virales , Endopeptidasas/genética , Endopeptidasas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Escherichia coli/genética , Escherichia coli/virología , Regulación Viral de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Codón Iniciador/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ADN Viral/genética , Bacteriófagos/genéticaRESUMEN
Bacterial oligopeptide transporters encoded by arrays of opp genes are implicated in a wide variety of physiological functions including nutrient acquisition, cell-to-cell communication, host-pathogen interaction. Combining the five opp genes in one oppABCDF operon of Escherichia coli assumes unified principle of their transcriptional regulation, which should provide a comparable amounts of translated products. This, however, contradicts the experimentally detected disproportion in the abundance of periplasmic OppA and the trans-membrane subunits OppB and OppC. As a first step towards understanding differential regulation of intraoperonic genes we examined genomic region proximal to oppB for its competence to initiate RNA synthesis using in silico promoter predictions, data of high-throughput RNA sequencing and targeted transcription assay. A number of transcription start sites (TSSs), whose potency depends on the presence of cationic oligopeptide protamine in cultivation medium, was found at the end of oppA and in the early coding part of oppB. We also show that full-size OppB conjugated with EGFP is produced under the control of its own genomic regulatory region and may be detected in analytical quantities of bacterial cell culture.
Asunto(s)
Biología Computacional , Escherichia coli/genética , Perfilación de la Expresión Génica/métodos , Ensayos Analíticos de Alto Rendimiento , Biología Computacional/métodos , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genes Reporteros , Proteínas de Transporte de Membrana/genética , Oligopéptidos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Sitio de Iniciación de la TranscripciónRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Expresión Génica , Genes Reporteros , Vectores Genéticos/genética , Regiones Promotoras Genéticas , ADN Superhelicoidal , Regulación Bacteriana de la Expresión Génica , Reproducibilidad de los Resultados , Transcripción GenéticaRESUMEN
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.