Kinetics of Riboswitch Regulation Studied By In Vitro Transcription

Riboswitches encompass messenger ribonucleic acid transcripts that sense the concentration of small molecule metabolites through binding the target compound and then control the expression of metabolite-related genes in response to the metabolite concentration. While much of the riboswitch-related research has focused on the remarkable ability of different aptamer domains to adopt the intricate structures required to bind a spectrum of biological metabolites with high affinity and specificity, less attention has been paid to the mechanism of riboswitch action. Specifically, the genetic control element of the riboswitch, known as the expression platform, must function cotranscriptionally in the case of transcription termination-controlled riboswitches. By correlating the transcriptional kinetics of the entire switch and the kinetics and thermodynamics of metabolite binding of the aptamer domain, it was found that the FMN-binding riboswitch in the 5′ UTR of the Bacillus subtilis ribGBAHT operon functions as a kinetically controlled genetic switch chiefly dependent upon transcriptional pausing and the concentration of the target metabolite. This study has emphasized the importance of studying the switch in its entirety and in the context of an actively transcribing RNA polymerase. Herein I will describe the study of the kinetics of riboswitch transcription and the proposed mechanism for the transcription termination-associated riboswitch control of riboflavin-related genes.