Quantification of Growth-Controlled Genes Transcription Dynamics by Live, Single-Cell Imaging
A common assumption in biology is that a transcription factor affinity for a promoter sequence, and thus the target promoter transcriptional output is largely dictated by its binding sequence. However, the exact relationship between promoter sequence and transcriptional output is not completely understood. As an example, the Shore lab observed a very low sequence similarity in between the 138 different Ribosomal Protein Genes (RPG) promoters constituting the major growth regulon in yeast, despite the fact that they share a common set of transcription factors (Knight et al., Genes and Development 2014). These housekeeping genes are responsible for 60% of the mRNA produced in rapidly growing cells and thus for a considerable energy cost to the cell. Therefore this promoter family offers a unique opportunity to understand how different promoter architectures can result in a similar expression output.
In this project, we aim at monitoring transcription arising from single RPGs in individual live yeast cells in controlled environmental conditions and specific genetic backgrounds, to assess the direct influence of the promoter sequence, TF binding and nucleosome positioning. To achieve this, we will use a single-mRNA in vivo probing method: the PP7 system (Larson et al., Science 2011). This technique will enable us to calculate the on/off rate and elongation speed from specific single RPGs promoter in various growth conditions and TF mutants. These measurements will allow us to gain insight into noise in housekeeping gene expression and assess the transcriptional mode of expression: bursting or constitutively expressed. Combining these single-cell measurements to the cell population studies information gathered by the Shore lab, we will be able to characterize both qualitatively and quantitatively the regulation of growth-controlled genes, such as RPGs.
Schlagworte: Single-cell, transcription, RPG, yeast, monitoring, growth, microscopy, PP7, TORzurück