Gene expression is comprised of many successive stochastic interactions between various cellular components (promoters, polymerases, etc.). The outcome of this is a phenotypic variability between cells in a population which has significant physiological consequences in natural and synthetic systems. One noteworthy source of noise in gene expression is fluctuations in promoter activity. However, precise characterization of these fluctuations has not yet been established.
Research into transcriptional dynamics is largely based on two complementary imaging approaches: SM-FISH measurements which cannot directly probe temporal effects, and live tracking of large RNA cassettes that are typically composed of 24 binding sites for RNA binding protein-fluorescent protein (RBP-FP) fusions. However, live tracking of single molecules in single cells is often not possible due to limitations of the RBP binding cassette such as cassette size and fluctuations in RBP occupancy, which camouflage successive transcriptional events.
Using novel RBP-FP cassettes designed in our lab we can track dynamically single mRNA molecules tagged with cassettes containing only four binding sites for PP7 coat protein (PCP) or Qβ coat protein (QCP). These short cassettes are stably bound and allow us to measure transcriptional events at high resolution. We implemented these cassettes by installing them at the transcriptional start site (TSS) of a T7-promoter in E. coli. Our data reveals that the T7 promoter can generate transcription bursts of one, two, three, or four RNA molecules. In addition, our data shows that we can resolve individual degradation events of single RNA molecules, which surprisingly also seem to be detected in bursts.
Our new cassettes allow us to explore the dynamics of transcription in unprecedented temporal resolution that may lead to a deeper understanding of transcriptional processes in bacteria.