Universal Actuator for Efficient Silencing of Genes Based on Convergent Transcription Resistant to Rho-Dependent Termination.

Dynamic control is a distinguished strategy in modern metabolic engineering, in which inducible convergent transcription is an attractive approach for conditional gene silencing. Instead of a simple strong "reverse" () promoter, a three-component actuator has been developed for constitutive genes silencing. These actuators, consisting of promoters with different strengths, the ribosomal transcription antitermination-inducing sequence -AT, and the RNase III processing site, were inserted into the 3'-UTR of three metabolic genes. Second and third actuator components were important to improve the effectiveness and robustness of the approach. The maximal silencing folds achieved for , , and were approximately 7, 11, and >100, respectively. Data were analyzed using a simple model that considered RNA polymerase (RNAP) head-on collisions as the unique reason for gene silencing and continued transcription after collision with only one of two molecules. It was previously established that forward () RNAP with a trailing ribosome was approximately 13-times more likely to continue transcription after head-on collision than untrailed -RNAP which is sensitive to Rho-dependent transcription termination (RhoTT). According to the current results, this bias in complex stabilities decreased to no more than (3.0-5.7)-fold if RNAP became resistant to RhoTT. Therefore, the developed constitutive actuator could be considered as an improved tool for controlled gene expression mainly due to the transfer of -transcription into a state that is resistant to potential termination and used as the basis for the design of tightly regulated actuators for the achievement of conditional silencing.