Triplex Modulation of Artificial Transcription Units

Abstract In cells, gene expression regulation is affected by a complex network of many factors. Although the interaction of proteins with genomic regulatory portions is considered the main regulatory mechanism, formation of nucleic acid triple-helical structures (i.e., triplexes) in the proximity of promoters was recently demonstrated to modulate transcription either with inhibitory or enhancing effects on the transcription process. In this study, hybrid DNA-RNA triplex structures identified in E. coli genome were characterized for their specific geometry-dependent effects on transcription regulation in vitro. Promoter sequences sraGp, safAp, lrpp, and poxBp1 were selected using Triplexator software and a set of specific parameters (e.g., type of promoter architecture, length of triplex, and proximity to the promoter consensus). Initially, temperature-dependent denaturation analysis and electrophoretic mobility shift assay (EMSA) were used for thermodynamic characterization of the triplex formation, while kinetic analyses of in vitro RNA polymerization from engineered transcription units were carried out to determine the modulatory effect. Rates of transcription were estimated using a fluorescent reporter, specifically, fluorogenic RNA aptamer Broccoli, which was integrated in the engineered transcription units as the actively transcribed product. In addition, the engineered transcription units integrated: σ70 bacterial promoter consensus from E.coli, and sequences that could form triplex structures in the presence of the associated single-strand RNA. In these conditions, the tested transcription units showed predictable transcription inhibition or enhancement, compared to a reference that did not contain the triplex-forming RNA, in line with previously reported results. The hybrid triplex nanostructures were used to confirm that genetic regulatory effects could be achieved using bacterial sequences, suggesting that this phenomenon might be a new general paradigm of gene expression regulation in cells.