Dissection of the Rate Constants of a Transcription Repression Mechanism from Live Single Cell, Single Molecule Microscopy Data

Transcription is a critical process in cells, as it allows to transform the information stored in the DNA and shaped by evolution, into RNA molecules that, once translated into proteins, are capable of performing a multitude of tasks that are necessary for maintain-ing the cell alive. Aside from identifying the main molecules involved in transcription, to fully compre-hend this process, we need to characterize its dynamics. This will allow a better under-standing of the mechanisms regulating gene expression. The regulatory mechanisms of gene expression are the means by which cells activate or repress, fully or to some extent, a gene’s transcriptional activity. It is this regulation that makes possible the response to environmental changes, as well as the establishment of critical internal cycles, such as the cycle responsible for cell replication. Here, we investigated, at the single cell, single gene level, the dynamics of the process of transcriptional regulation the promoter LacO3O1 by gene-specific regulatory mole-cules, namely, inducers. Our goal was to, from live, single cell, single molecule data, obtain the values of the rate constants associated with the repression mechanism of tran-scription of this promoter. Based on direct measurements of RNA production kinetics at different induction levels, and by estimating the RNA production rate at infinite induction we inferred that, under full induction, the LacO3O1 promoter, on average, spends 12% of the time between consecutive RNA productions in the OFF state