Transcript counting in single cells reveals dynamics of rDNA transcription

Abstract

Abstract Most eukaryotes contain many tandem repeats of ribosomal RNA genes of which only a subset is transcribed at any given time. Current biochemical methods allow for the determination of the fraction of transcribing repeats (ON) versus non‐transcribing repeats (OFF) but do not provide any dynamical information and obscure any transcription activity at the single‐cell level. Here, we use a fluorescence in situ hybridization (FISH) technique that allows the detection of single‐RNA molecules in individual yeast cells. We use this method complemented with theoretical modeling to determine the rate of switching from OFF to ON (activation rate) and the average number of RNA molecules produced during each transcriptional burst (burst size). We explore how these two variables change in mutants and different growth conditions, and show that this method resolves changes in these two variables even when the average rDNA expression is unaltered. These phenotypic changes could not have been detected by traditional biochemical assays.

Keywords

• rDNA
• single‐molecule FISH
• Rpd3