Dynamics of Open States and Promoter Functioning in the appY_red and appY_green Genetic Constructions Based on the pPF1 Plasmid

Abstract

Background: Although the role of dynamic factors in DNA function still remains unclear, research in this direction is a rapidly developing area of molecular biology. In this work, the genetic constructions appY_red and appY_green, based on the plasmid pPF1 and containing a fragment of Escherichia coli (E. coli) DNA with predicted promoter-like regions, are considered complex dynamic systems in which local sites of double helix unwinding, called open states, can arise and propagate. The purpose of the article is to show the existence of a connection between the dynamics of open states and the functioning of predicted promoters. Methods: We experimentally verified the functionality of the predicted promoters using a reporter vector. Using a reverse transcription reaction, transcription start sites were identified indicating the presence of two divergent promoters, one on each strand. In mathematical studies, a dynamic model was used that described open states as one-soliton solutions (kinks) of a system of nonlinear partial differential equations, and the influence of the torque Mτ on the dynamics of kinks was taken into account. Results: Fluorescence analysis of colonies of E. coli cells transformed with plasmid constructions showed that one of the two promoters is stronger than the other and that the strength of the promoters depends on the orientation of the fragment under study in the plasmid. On the other hand, using mathematical modeling, the energy profiles of genetic constructions were calculated and the kink trajectories were constructed. In addition, by studying the effect of torsion moment in model studies, we found threshold torque values at which the behavior of kinks changes dramatically: from oscillatory to translational motion. The minimum values of torsion moment required to initiate the transcription process were also assessed. Conclusions: A comparative analysis of the results of experimental data and model calculations showed a good correlation between the preferred starting points and the direction of transcription, which in turn confirmed the existence of a relationship between the dynamics of open states and the functioning of promoters.

Keywords

• open states
• ppf1 plasmid
• e. coli appy gene
• transcription
• mathematical model